Dietary Biotin Effects on Polyunsaturated Fatty Acids in Chick Tissue Lipids and Prostaglandin E2 Levels in Freeze-Clamped Hearts

Dietary Biotin Effects on Polyunsaturated Fatty Acids in Chick Tissue Lipids and Prostaglandin E 2 Levels in Freeze-Clamped Hearts B. A. W ATKINS1'2 and F. H. KRATZER Department of Avian Sciences, University of California at Davis, Davis, California 95616 (Received for publication December 8, 1986)

1987 Poultry Science 66:1818-1828 INTRODUCTION

The metabolic disorder occurring in broiler chickens, fatty liver and kidney syndrome (FLKS), has been described by several investigators (Marthedal and Vellinge, 1958; Hemsley, 1965; Riddell etal., 1971; Whitehead etal., 1975; Lohr, 1975). Deficiency of dietary biotin has been implicated in the development of FLKS (Roland and Edwards, 1971) and the syndrome can be prevented with biotin supplementation (Whitehead and Blair, 1974; Payne etal, 1974; Bannister etal., 1975). The significant accumulation of lipid observed in liver and kidney tissues from FLKS is primarily triglyceride and the composition typically contains elevated palmitoleate and reduced stearate (Whitehead, 1975). Johnson et al. (1972) suggested that a high level of liver palmitoleate is a good indicator of FLKS. Studies on the metabolic changes that develop during FLKS (Bannister, et al., 1975; Bannister, 1976) demonstrated a dramatic decrease in pyruvate carboxylase (PC) activity and, therefore, lowered gluconeogenesis (Bannister et al., 1975; Pearson et al., 1976). Also, higher

Present address: Department of Poultry Science. Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. 2 To whom correspondence should be addressed.

lipogenesis in liver tissue was observed in chickens fed a low biotin diet (Hood et al., 1976). A marginal deficiency of dietary biotin in chickens was found to have differing effects on PC and acetyl-CoA carboxylase (ACC) activities during FLKS (Pearson et al., 1976). Biotin deficiency, demonstrated by low liver biotin (<.80 |xg/g), significantly reduced PC activity; however, ACC activity was elevated during mild symptoms of FLKS and depressed with severe FLKS. Biotin deficiency may also be involved in the Acute Death Syndrome (ADS) that affects chickens (Kratzer et al., 1985), as liver biotin levels were significantly reduced in several chickens that died of ADS. Many of the ADS symptoms and gross pathological findings resemble FLKS (Ononiwu et al, 1979; Steele et al., 1982). Hulanetal. (1980) found that dietary d-biotin was beneficial in reducing ADS mortality. However, the cause of ADS is still controversial and the role of biotin in this syndrome is not clear. Fogerty et al. (1984) suggested a correlation between the Sudden Infant Death Syndrome (SIDS) in infants and FLKS in broiler chickens. The biotin content in the livers of SIDS victims was significantly lower than in livers from infants who died of other causes (Johnson et al., 1980; Heard etal, 1983; Fogerty etal, 1984). Liver lipids from SIDS victims had a lower level

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ABSTRACT Chicks were fed a purified diet with 0, 100, 200, 300, 400, or 500 n-g/kg diet of added biotin to determine the effects of biotin deficiency on polyunsaturated fatty acids in tissue lipids. Body weight was reduced by biotin deficiency and liver and heart biotin levels varied with the biotin in the diet. Fatty acids in liver and lung from biotin-deficient chicks at 15 days contained elevated (P<.03) 18:3<«3 and 18:2co6 but prostaglandin precursors 20:3o)6 and 20:4co6 were reduced (P<.03) in liver lipids. Heart tissues from 15-day-old chicks fed the biotin-deficient diet were low (P<.03) in 20:3co6. Fe^djng^acetyjsalicylic acid in diets containing added biotin. (0, 100, 400, and 500 u.g/kg) did_noX^ignrficantly alter fatty acid levels in chick_tissjjg_lirjids-J)ut-srgnificaBtly.jeduced plasma prostaglandin E7 (PGE2). BiotiiTdeficiency reduced heart PGE2 levels in 22-day-old chicks. An 8-h fast reduced (P<.04) 20:4
BIOTIN EFFECTS ON POLYUNSATURATED FATTY ACIDS

on fatty acid metabolism in broiler chicks fed purified diets. Prostaglandins (PGE2 and 6KPGF) were measured in chick plasma and heart tissues to observe changes in these products derived from 20:4w6. MATERIALS AND METHODS

Animals and Diets. Day-old male broiler chicks were wingbanded, weighed, randomized, and placed in cages. Diets and water were provided ad libitum until birds were subjected to a fasting period or termination of the experiment. Each dietary treatment consisted of three pens of eight chicks each, reared in a temperature controlled battery brooder with raised wire floors. The basal diet contained all nutrients required for chicks (National Research Council (NRC), 1984) except available biotin (Table 1). TABLE 1. Basal diet to produce biotin in chicks Ingredient

deficiency

Amount (g/kg)

Isolated soy protein Corn starch Soybean oil1 Dried egg albumen 2 DL-Methionine CaC0 3 CaHP04-2H20 Mineral mix 3 Vitamin mix (biotin free)4 Cellulose

250.0 563.0 50.0 30.0 7.5

13.0 22.0 24.5 10.0 30.0

Total

1,000.0

Calculated composition Metabolizable energy, kcal/kg Crude protein, % Available biotin, jug/kg

3,560 23 0

'Soybean oil composition: 54% 18:2u;6; 22% 1 8 : 1 C J 9 ; 10% 16:0; 8.6% 18:3w3. 2 Analyzed avidin binding of dried egg albumen was 7.25 units/g (1 unit binds 1 microgram of biotin). 3

Provided in milligrams per kilogram of diet: C o ( C 2 H 3 0 ) - 4 H 2 0 , 20; C u S 0 4 - 5 H 2 0 , 97; FeSO„ • 7 H 2 0 , 640; K I 0 3 , 9; K2 HPO„, 4,950; KC1, 2,970; MgSO„-7H 2 0, 5,500; MnSO„-H 2 0, 297; NaCl, 9,900; N a 2 M o 0 4 - 2 H 2 0 , 9; Na2 S e 0 3 - 5 H 2 0 , .66; ZnO, 120. 4 Provided in milligrams per kilogram of diet (except as noted); vitamin A, 4,500 IU; vitamin D 3 , 500 ICU; vitamin E, 50 IU; menadione, 1.5; thiamine, 15; riboflavin, 15; niacin, 50; calcium pantothenate, 20; pyridoxine, 6; folic acid, 6.0; vitamin B 1 2 , 20 Mg; choline chloride (70%), 2,000; butylated hydroxytoluene, 200.

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of 20:3w6 than liver lipids of non-SIDS victims, suggesting a defect in essential fatty acid (linoleate) metabolism. Prostacyclin I2 (PGI2) is a major product of arachidonic acid (20:4w6) metabolism in the endothelial cells of blood vessels that acts as a potent inhibitor of platelet aggregation (Moncadaetal., 1977;Moncadae? al., 1978;Needleman et al., 1979). The PGI2 is synthesized in response to thromboxane A2 (TXA2) production by platelets (Johnson et al., 1983). These two eicosanoids derived from 20:4w6 have potent yet opposite effects. The PGI2 facilitates dilation of blood vessels and inhibits platelet formation, whereas TXA2 induces platelet aggregation and causes vasoconstriction. The PGI2 is unstable and has a short biological half-life in blood (Johnson et al., 1983). A relatively stable metabolite of PGI2 is 6-keto-PGF r a (6KPGF), which has been measured frequently in plasma as an index of PGI2 formation in human subjects. Vermylen et al. (1981) showed that a reduction of serum thromboxane B 2 (TXB2) levels was associated with an increase in 6KPGF in plasma. Other prostaglandins (PG) have been measured in plasma of humans and animals as well (McCosh et al., 1976). Prostaglandin E2 (PGE2), also a product of 20:4to6, causes vasodilation of blood vessels and lowers blood pressure (Johnson et al, 1983). The action of both PGI2 and PGE2 on vascular tissue is quite similar in heart tissue although PGI2 is more potent at reducing blood pressure (Armstrong et al., 1978). The measurement of PGE2 in canine plasma and myocardial homogenates was reported by McCosh et al. (1976). Claeys et al. (1981a) reported high levels of PGE2 synthesized from arachidonic acid in isolated chicken aorta. The PGE2 appears to be an important metabolite of 20:4co6 and has a major role in homeostasis in the chicken, as PGI2 is not synthesized in the aorta (Claeys et al., 1981b). Bult et al. (1981) found that PGE2 suppresses aggregation of avian thrombocytes, whereas PGI2 showed little antiaggregatory activity. It appears that in avian species PGE^isan important prostaglandin in regulatings vascular control in heart tissue. The effects of biotin deficiency on fatty acid metabolism might result in reduced prostaglandin fatty acid precursors that would cause lowered PG biosynthesis. Arachidonic acid was reported to be reduced during biotin deficiency in chicks (Pearson et al., 1976; Kratzer et al., 1985; Watkins and Kratzer, 1987). This study was performed to investigate the effects of biotin

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WATKINS AND KRATZER

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Collection and Preparation of Samples. Chicks were anesthetized in 20 s with Halothane (Halocarbon Laboratories, Inc., Hackensack, NJ) vapors prior to collection of blood and heart tissues for prostaglandin assay. Blood was obtained from the right atrium of the heart with a syringe rinsed with a 4.5-mM ethylenediaminetetraacetate (EDTA) solution containing aspirin at 444 mg/L. Blood was transferred to a polypropylene tube containing .09 mL/mL of blood of the EDTA/aspirin solution. Tubes were centrifuged at 1,500 x g for 10 min at 22 C and the plasma was removed to polypropylene tubes, frozen, and stored at-196 C. Plasma was collected from 8 and 12-day-old chicks. Heart samples were collected immediately from anesthetized chicks by exposing the thoracic cavities and freeze-clamping the tissue between aluminum plates, cooled to -196 C with liquid nitrogen. Hearts were collected from 22-day-old chicks and stored at -196 C. Tissues used for fatty acid and biotin analyses were removed from chicks after killing by cervical dislocation and placed on ice, frozen, and stored at -20 C. Tissue samples were collected from chicks at different ages to determine when biotin deficiency alters polyunsaturated fatty acid (PUFA) composition in tissue lipids. Some chicks were

TABLE 2. Performance characteristics and incidence of deficiency symptoms of chicks fed or biotin-adequate diets with and without aspirin for 20 days1 Biotin/aspirin treatment2

Mortality

(Mg/kg) 0 100 200 300 400 500 0(SM) 0 + ASA 100 + ASA 4 0 0 + ASA 5 0 0 + ASA 0 (SM) + ASA a

c

Weight gain (X)

Feed:gain

(no. chicks)

(g)

(g/g)

1 0 0 0 0 0 0 3 3 0 0 0

200 228 387 361 343 326 356 187 247 334 364 387

± + ± ± + ± + + + + ± ±

58b 79b 108* 137* 123* 112* 143* 53b 75b 67* 96* 109*

1.69* 1.53bc 1.50 c 1.43c 1.46 c 1.49 c 1.69* 1.65* b 1.63* b 1.47 c 1.50 c 1.62ab

biotin-deficient

Perosis score 3

Dermatitis score 3

2.50 1.90 .13 .13 .10 0 .12 2.70 2.00 .12 .13 .11

2.3 1.7 .3 0 0 0 0 2.4 1.0 0 0 0

Values in columns with different superscripts are significantly different (P<.05).

1

Values are means ± standard deviations, triplicate lots of eight chicks per treatment.

2

ASA = acetylsalicylic acid fed at 500 mg/kg of diet; SM = stockmash diet (control).

3

Values are mean scores: 0 = normal; 4 = severe perosis or dermatitis.

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d-Biotin (United States Biochemical Corporation, Cleveland, OH) and acetylsalicylic acid (aspirin, Sigma Chemical Co., St. Louis, MO) were added to the basal diet as shown in Table 2. Aspirin was added to reduce prostaglandin synthesis during biotin deficiency to observe any aggravation of symptoms. A stockmash diet (containing corn and soybean meal) was used as a control. The diet contained 150 fig biotin/kg and 5% crude fat with the following fatty acid composition: 18:2co6, 47%; 18:lo>9, 28%; 16:0, 15%; and 18:3w3, 1.8%. Chick body weights and feed consumptions were measured and the experiment continued for 20 days. During tissue collection, chicks were humanely killed by cervical dislocation or with a general anesthetic. Chick tissues were sampled at 15 and 20 days of age to determine the effects of biotin on fatty acid composition and PG levels. Biotin Deficiency Symptoms and Scoring. Perosis, characterized by an enlargement and twisting of the hock joint, was observed for chicks during the experiment at 21 days and scored from 0 to 4 according to severity. Footpad dermatitis was also observed at 21 days and scored with a value from 0 to 4. Many chicks that died showed the "flip-over" syndrome, characterized by being found dead, lying on their backs with neck extended.

BIOTIN EFFECTS ON POLYUNSATURATED FATTY ACIDS

with chloroform:methanol (2:1, v/v) prior to packing, and each column contained approximately 6.5 g of packing material. The analysis of methylated fatty acids was performed with Hewlett-Packard 5700A and 5710A gas chromatographs (GC) operated with dual columns and flame ionization detectors. Detector gases were hydrogen and compressed air. The nitrogen carrier gas flow was adjusted to 20 mL/min and isothermal operation of the GC was performed at 180 C with a sensitivity of 10 and attenuation of 2 x . Both GC were equipped for use with a 3352A laboratory data system on a 2100 computer (Hewlett-Packard, Santa Clara, CA). A 7671A autosampler was used with the 5700A GC for automatic injection of samples. All injections for GC fatty acid analysis contained 2 u,L of the methyl esters in isooctane. Fatty acid peaks were identified by injection of a standard mixture of methylated fatty acids prepared from a triglyceride mixture (Nu-Chek-Prep, Elysian, MN). Prostaglandin Determinations. The levels of PGE2 and 6KPGF were determined in plasma and freeze-clamped heart tissue of chicks. Plasma collected from chicks was acidified to pH 3.5 with 2/V HC1 for PGE2 and to pH 3.0 with 2M citric acid for 6KPGF before isolating the PG by liquid-column chromatography (Granstrom, 1979; Shrinka and Lucas, 1981). The extraction of PG from heart tissue was performed with ethyl acetate by a method similar to Hertelendy and Biellier (1978). Liquid column chromatography (LC) was performed with silica and C-18 columns (BondElut silica and C-18, Analytichem International, Harbor City, CA). Plasma PGE2 was isolated from the acidified plasma using a C-18 column (100 mg/1.0 mL) that was washed with methanol and then distilled water. The PGE2 was eluted with methanol, evaporated under N 2 gas at 22 C, and reconstituted with 1 mL of PGE2 radioimmunoassay (RIA) buffer. Plasma 6KPGF was isolated from acidified plasma in a two-step LC procedure. The sample was first applied to a C-18 column that was washed with methanol, then distilled water. The 6KPGF was then eluted onto a washed (5 mL of benzene :ethylacetate, 80:20, v/v) silica column (500 mg/2.8 mL) with 1 mL of ethyl acetate. A series of solvent mixtures containing benzene:ethyl acetate (60:40, v/v) and increasing methanol were applied to the silica column. Elution of 6KPGF from the silica column was done with 5 mL of benzene:ethyl acetate:methanol (60:40:30, v/v/v).

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fasted for 8 h to determine the effects of fasting on PUFA compositions in liver and heart tissues of 20-day-old chicks. Liver and heart tissues from 8-h fasted chicks were analyzed for biotin to observe the effects of fasting on biotin content. Biotin Analysis. Biotin in feed and chick tissues was determined by a method described by Hood (1975, 1977). The technique involved isotopic dilution of 14C-biotin by sample unlabeled biotin and the use of avidin as a binding protein for biotin. The d-(carbonyl-14C)-biotin (Amersham, Arlington Heights, IL) had a specific activity of 58 mCi/mM and the avidin (Sigma Chemical Company, St.Louis, MO) contained 12.00 units/mg. Tissues were homogenized for 3 min with 2N H 2 S0 4 and autoclaved at 121 C for 15 min to liberate biotin. Feed samples were treated in a similar manner but autoclaved for 60 min at 121 C. Scintillation vials containing the d-14Cbiotin from the biotin analyses and 5 mL of cocktail (3a70B, Research Projects International Corp., Prospect, IL) were vortexed and counted in a Searle Mark III, 6880 Liquid Scintillation System (Searle Analytic, Inc., Des Plaines, IL). The avidin-binding capacity of dried egg albumen used in the basal diet was determined by a reverse application of the biotin analysis procedure. One gram of dried egg albumen was used in the biotin assay for 0, 100, 200, 300, and 400 |J.L of the dried egg albumin solution. The assay was performed in triplicate. Lipid Analysis. Lipids from all tissues analyzed for fatty acid composition were extracted by modifying the methods of Folch et al. (1957) and Bligh and Dyer (1959). Methyl esters were prepared by esterification with 12% boron trifluoride in methanol (Metcalfe and Schmitz, 1961; Metcalfe et al, 1966). Esters were then extracted with 1 mL of isooctane and washed with 8 mL of triple distilled water. Verification of complete esterification was determined by thin-layer chromatography (TLC) of the methyl esters on Silica Gel G plates (Sigma Chemical Company, St. Louis, MO). The TLC plates were developed in a solvent system containing petroleum ethendiefhyl ethenglacial acetic acid (90:10:1, v/v/v). Separation of methylated fatty acids was performed by gas-liquid chromatography with a 3.0-m by 2.67-mm (id) stainless steel column, packed with 10% SP-2330 on 100/120 chromosorb W-AW (lot no. T21773, Supelco Inc., Bellefonte, PA). Column tubing was rinsed

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WATKINS AND KRATZER

ment differences. All analyses were performed on a VAX-11/780 Alcor computer and used SAS statistical programs (SAS Institute Inc., 1983). When significant F values were obtained, differences in treatment means were determined by Tukey's or Bonnferonni's methods (Neter and Wasserman, 1974). RESULTS AND DISCUSSION

Mortalities, body weights and gains, feed conversions, and deficiency symptoms of chicks fed the various dietary biotin regimens are shown in Table 2. Mortality was greatest for chicks fed 0 or 100 (jug of biotin. Addition of 200 (jug of biotin to the basal diet resulted in maximum growth; this level of biotin was close to the requirement of 150 |xg/kg for the chick (NRC, 1984). Feed conversion improved with higher dietary biotin. The severity of perosis and dermatitis symptoms decreased with increasing biotin. The addition of aspirin to diets resulted in higher mortality in chicks fed 0 and 100 |xg of biotin/kg of diet. Body weight gains of chicks fed biotin at 200 |xg/kg of diet were not significantly different from chicks fed higher levels of biotin. Feeding stockmash to chicks did not improve body weight gains and aspirin did not depress weight gains. Feed conversion improved with the addition of biotin to the purified diets and when biotin levels were greater than 100 (xg/kg of diet, feed conversion was better (P<.05) than for birds fed the stockmash diet. Aspirin fed at 500 mg/kg of diet caused no change in feed conversion. Aspirin appeared to aggravate slightly the perosis scores in chicks fed 0 and 100 \ig of added biotin.

TABLE 3. Biotin content of 20-day-old chick liver and heart tissues1 Diet treatment

Biotin

Heart

Liver

(Mg/kg diet)

(ng/g wet tissue)

Fed

0 100 400 0 (Stockmash)

968 ± 1,227 + 3,659 ± 2,399 ±

91a lll c 202 a 92b

866: 67 L 1,081 : 73* 2,506 : 132 a 2,247 : 167 a

Fasted

0 100 400 0 (Stockmash)

916 ± 1,205 ± 3,792 ± 2,435 ±

108 d 95c 177 a 130b

776± 130 d 948 ± 1 2 1 c 2,583 ± 148 a 2,172 ± 9 6 b

a 1

Values in columns with different superscripts are significantly different (P<.01). Values are means ± standard deviations, five chicks sampled per treatment.

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The extracted 6KPGF was dried under N 2 gas at 22 C and reconstituted with 1 mL of 6KPGF RIA buffer. Heart PG were isolated using a silica column (Hillier and Dilley, 1974) that was washed with 2 mL of benzene:ethyl acetate (60:40, v/v). The prostaglandin A and B were eluted with 3 mL of benzene:ethyl acetate (60:40, v/v). The PGE were eluted with 6 mL of benzene:ethyl acetate:methanol (60:40:2, v/v/v), and PGF were eluted with 2 mL of benzene:ethyl acetate:methanol (60:40:40, v/v/v). Each fraction was evaporated under N2 gas in a 50 C water bath and reconstituted with RIA buffer. The recoveries of PGE2 and 6KPGF were measured from plasma and heart homogenate samples spiked with 3H-PGE2 and 3H-6-KPGF (New England Nuclear, Boston, MA) after LC. Approximately 91% of 3H-PGE2 and 88% of 3 H-6-KPGF were recovered. Levels of PGE2 and 6-KPGF were quantified by RIA from extracts of plasma and heart samples. Radioactivity in samples prepared from a PGE2 kit that used ,25 I-PGE 2 as the labeled antigen (NEK-020, New England Nuclear, Boston, MA) was counted with a Micromedic Systems gamma counter, model 4/200 (Micromedic Assay CompuCenter, Horsham, PA).The radioactivity from labeled 3H-6-KPGF in samples prepared from the 6-KPGF RIA kit NEK025 (New England Nuclear, Boston, MA) was counted by liquid scintillation as described earlier. Statistical Analysis. Data were subjected to either a one-way or two-way analysis of variance (Snedecor and Cochran, 1967) to detect treat-

BIOTIN EFFECTS ON POLYUNSATURATED FATTY ACIDS

in a previous experiment (Watkins and Kratzer, 1987). The present data indicate that changes in liver PUFA of biotin-deficient chicks occur before 21 days. Total fatty acids were measured in liver and heart tissues from ad libitum-fed and 8-h fasted 20-day-old chicks. Biotin deficiency resulted in elevated 16:1, 18:2w6, 20:0, and 18:3w3 and decreased 17:0, 20:3w6, 22:5w6, and 22:5w3 in the liver (Table 7). Fasting resulted in increased 18:2w6 and 18:3co3 and lowered 20:lw9, 20:2co6, and 22:4co6. Heart fatty acids (Table 8) measured from biotin deficient chicks showed elevated 16:0, 16:1, and tl8:2 and decreased 17:0, 18:0, 20:lw9, 20:2w6, and 20:3w6. During fasting, 17:0, 20:2w6, 20:4a>6, and 22:4co6 decreased whereas 18:lw9 increased in heart. Many fatty acids in liver and heart showed the same trends during biotin deficiency with elevated 16:1 and reduced 18:0, 20:lw9, and 20:3co6 at 15 days. Changes in lung tissue fatty acids were similar to liver changes during biotin deficiency. In lung, 16:1, 18:2w6, and 18:3w3 were increased as in liver and 18:0 and 20:lw9 were lowered. Delta-6 desaturase activity in liver microsomes of rats appeared to decrease with age (Brenner, 1974). If delta-6 desaturase activity is higher in young animals, chicks at 20 days of age may have sufficient enzyme activity to

TABLE 4. Mean weight percentage of total liver fatty acids of 15-day-old chicks fed biotin-deficient or biotin-adequate diets1

Fatty acid

0 jug/kg Biotin

400 Mg/kg Biotin

Stockmash (control)

16:0 16:1 18:0 18:lu>9 18:2co6 20:0

20.69 4.58 a 15.05 b 18.30 a 23.95 a ,40 a 1.16 a .llb

20.94 1.37b 20.75 a 10.92 b 18.02 b

20.61 1.61 20.03 13.96 16.24

18:3CJ3

20:lu;9 20:2u6 20:3u)6 20:4w6 22:4CJ6

22:6a>3 22:5u>6 20:5o;3

•36u

.83b 9.70 b .53b 2.90 b .36 b .88

13

K

.40 b .21a .42

1.70a 16.65 a .85 a 5.85 a .74 a .93

.10 .14 .37 .55

1.91 15.18 .99

6.03 1.21 .37

Pooled SEM

.90

1.05 1.90 2.07 .67 .05 .24 .02 .04 .16

1.50 .08 .58 .08 .11

ab ' Values in rows with different superscripts are significantly different (P<.03). 1

Mean values of four chicks per dietary treatment and pooled standard errors of the mean (SEM).

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Significant differences (P<.01) were found in the biotin content of chick livers and hearts (Table 3). The biotin content of chick livers reflected dietary biotin levels fed to chicks as did heart biotin levels. Heart biotin content showed a significant decrease with dietary deficiency (<100 fi-g/kg). Severe FLKS symptoms were observed in chicks that had liver biotin levels below 800 ng/g of tissue (Hood et al., 1976). Chicks fed a diet with no added biotin had liver biotin levels near 800 ng/g. An 8-h fast did not reduce biotin levels in liver or heart. Total fatty acids were determined in liver, heart, and lung tissues from ad libitum-fed 15day-old chicks. In liver tissue (Table 4) from biotin-deficient chicks, 16:1, 18:lw9, 18:2co6, 20:0, and 18:3w3 were elevated whereas 18:0, 20:lw9, 20:3w6, 20:4oo6, 22:4
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WATKINS AND KRATZER TABLE 5. Mean weight percentage of total heart fatty acids of 15-day-old chicks fed biotin-deficient or biotin-adequate diets1

0 Mg/kg Biotin

400 Mg/kg Biotin

Stockmash (control)

16:0 16:1 18:0

19.73 3.89 a 13.25 b 21.52 28.26

17.86 2.29 b 17.30 a 20.14 26.64

18.52 3.01 18.49 21.13 21.83

18:1CJ9 18:2GJ6

20:0 18:3u;3 20:lcj9 20:2o>6 20:3^6 20:4CJ6 22:4CJ6 22:66

20:5o>3

.10

1.24 .24 b .37

1.06 b 8.97 .54 .20 .14 .50

Pooled SEM

1.02 .83

1.50 2.12 .86

.009

a

.09 .20 .54 .47

1.66a 10.59

2.06 12.63

1.66

.93 .46 .34 .48

.15 .05 .04 .07

.09 .84

.35 .39

.85 .26 .20 .39

.17 .02 .05 .21

a ' b Values in rows with different superscripts are significantly different (P<.03). 1

Mean values of four chicks per dietary treatment and pooled standard errors of the mean (SEM).

convert linoleate to arachidonate even with a diet marginal in biotin (moderate elongation of 18:3o)6). The reduction in 20:3w6 and 20:4w6 at 15 days may result from the need of liver to supply other tissues with 20:4w6 where the enzymes for converting linoleate to arachidonate are not fully active or absent. Vessel endothelial

cells and platelets are dependent upon liver to supply 20:4u>6 for PG synthesis. Prostaglandin levels were determined in plasma and heart (Table 9). In 12-day-old chicks, both plasma PGE2 and 6KPGF (stable metabolite of PGI2) were elevated in biotin-deficient chicks.When aspirin was fed, PGE2 was

TABLE 6. Mean weight percentage of total lung fatty acids of 15-day-old chicks fed biotin-deficient or biotin-adequate diets1

Fatty acid

0 Mg/kg Biotin

400 Mg/kg Biotin

Stockmash (control)

16:0 16:1 18:0 18:lco9 18:2co6 20:0 18:3u>3

31.14 3.84 a 12.22 b 17.85 16.05 a

31.56 2.86 b 13.37 a 19.09 13.29 b

29.00 2.98 12.59 21.73 12.54

20:1CJ9

20:2o;6 20:3^6 20;4co6 22:4w6 22:6u>3 22:5u>6 20:5CJ3

.48

.44

.24 .18 .59 .39

1.82 10.27 1.73 1.15

2.30 10.92 1.89 1.15

2.26 11.21 1.78 1.34

.48 .99

.79 .88

.60 .57

.29

.71a .37 b

.27

.53b .46 a

Pooled SEM

1.44 .29 .43 .78 .57 .01 .06 .02 .02 .26 .54 .10 .10 .20 .06

a,b Values in rows with different superscripts are significantly different (P<.03). 1

Mean values of four chicks per dietary treatment and pooled standard errors of the mean (SEM).

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Fatty acid

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BIOTIN EFFECTS ON POLYUNSATURATED FATTY ACIDS TABLE 7. Mean weight percentage of total liver fatty acids of fed and fasted 20-day-old chicks fed biotin-deficient or biotin-adequate diets'

Pooled Fatty acid

Fed

Fasted

0 Mg/kg Biotin

100 Mg/kg Biotin ,

23.10 4.53 .31

.26

15.59 17.44

13.09 19.50

.03

18.45 b

20:2CJ6 20:3UJ6

20:4u)6 20:5u>3 22:4u>6

20.91 5.71

.06

22.16*

.22

.32

.83b .22* .56* 1.42 9.42

1.47* .17 b .35 b 1.27 8.83

.60

.74*

•

7 3

Kb

.47

22:5OJ6

.58

.35

22:6w3

2.47

2.82

22:5OJ3

.73

.83

23.29* .41* 1.50*

22.42* .26* b 1.51*

.17

.18 .48

• 4 6 ub .86 8.54

.94 7.30

.62

.63

• 6 3 ub .24 2.28* b .53b

1.69 1.30 .06

2.17 2.86 .04

.08

.03

.04

21.41 2.47 b .47* 18.09* 16.41

•_, . „ , s

23.71 7.07* .21b 11.48 b 19.33

20.88 5.82* b .18 b 13.45ab 19.66

SEM

15.21 b .14 b .44 b

1.05 .05 .21 .02 .08 .34

.24 .42 b

• 5 7 ub ,32* 1.99 b .80* b

2.24* 11.53

2.04

.74 .62

.82* 3.68* 1.02*

.10 .15 .21 .64 .16

a ' b Values in rows (fed/fasted or biotin treatments) with different superscripts are significantly different (P<.04). 1

Mean values of five chicks per dietary treatment and pooled standard errors of the mean (SEM).

TABLE 8. Mean weight percentage of total heart fatty acids of fed and fasted 20-day-old chicks fed biotin-deficient or biotin-adequate diets1 Pooled Fatty acid

Fed

Fasted

100 Mg/kg Biotin

0 Mg/kg Biotin /

16.97 2.12 1.19 b 16.41 17.13*

16:0 16:1 17:0 18:0 18:1U)9 tl8:2

17.38 1.47 1.43* 17.79 12.73 b .04

.03

18:2OJ6

27.14

28.22

.08 .68 .25

.08 .72 .26

20:0 18:3CJ3

20:lo>9 20:2^6 20:3u)6 20:4u;6 20:5u>3 22:4u6 22:5u>6 22:6a>3 22:5u)3

.71* 1.58 15.78* .44

1.02* .25 .52 .64

.51b 1.33 12.18 b .53

.74 b .21 .37 .58

18.00* 2.50* 1.12 b 15.65 b 15.73 .04* on nn^~ .08 .71

.21b .52 b 1.05 b 13.79 .44 .81 .22 33ab

.60

400 Mg/kg Biotin

SEM

- i

1724ab ab

1.80 1.13 b 16.98 a b 13.96 .04* 29.09* .09 • 6 8 Kb

16.29 b 1.09 b 1.68* 18.67* 15.10 .03b 26.18 b .08 .72

.23 .64* b 1.54*b 14.26

.33* .67* 1.77* 13.90

.49 .88

.53 .96 .27

• 1 9 Kb

.25

.75*

.47

.75

.51 .49 .13 .91

1.48 .03 .96

.004 .10 .01 .05 .21

1.46 .06 .10 .05 .17 .13

*' Values in rows (fed/fasted or biotin treatments) with different superscripts are significantly different (P<.04). 1

Mean values of five chicks per dietary treatment and pooled standard errors of the mean (SEM).

Downloaded from http://ps.oxfordjournals.org/ at Rutgers University Libraries/Technical Services on April 11, 2015

16:0 16:1 17:0 18:0 18:lu>9 tl8:2 18:2o;6 20:0 18:3^3 20:lu>9

400 jug/kg Biotin

WATKINS AND KRATZER

1826

TABLE 9. Prostaglandin levels (PGE2 and 6-keto-PGF, -a) in plasma and heart tissues of chicks fed biotin-deficient or biotin-adequate diets1 Heart, pg/g wet tissue

Plasma, pg/mL Biotin t r e a t m e n t

2

6-keto-PGF,-a

PGE2

± 143.8 ± 28.7 ± 12.9 ± 21.5 + 12.2

353.3 94.4 37.2 42.3 153.6

21.8 ± 4.3 101.9 ±12.8 97.5 ± 18.1

5266.7 ±539.4 4981.5 ± 977.3 5178.5 ±226.1

41.4 ± 7.1

4794.7 ± 712.2

± ± ± ±

191.0+ 506.3 ± 419.3 ± 241.0 ±

PGE2

(22-day-old)

(Mg/kg)

0 + ASA 100 + A S A 500 + ASA 0 (SM) + A S A

161.7 51.4 78.3 32.2 88.0

7.7 6.6 4.9 4.6

1.8 2.1 1.5 1.1

+ 100.4 ± 28.3 + 22.8 ± 18.8 ± 21.6

43.3 32.3 54.6 49.5

1 Values are means ± standard deviations for three chicks each (8-day-old and 12-day-old) and four chicks each (22-day-old) per dietary treatment. 2

SM = Stockmash diet (control); ASA = acetylsalicylic acid fed at 500 mg/kg of diet.

significantly depressed in plasma of 8-day-old chicks. Aspirin did not reduce 6KPGF in plasma. Heart PGE2 was found to be greatly lowered in biotin-deficient chicks (21.8 ± 4.3 pg/g). No difference in chick heart 6KPGF was observed among dietary biotin treatments. Chronic dosing of aspirin (20 to 30 mg/day) in human subjects (18 to 37 yr) was reported to significantly reduce platelet-formed TXB2 but had no significant effect in reducing 6KPGF levels in urine (Patrignani etal., 1983). Willems et al. (1982) found that pretreatment of human endothelial cells with aspirin (10 |xM) would inhibit cyclooxygenase (CO), but the activity of CO could be restored within 24 h to control levels after washing the cells and reincubating. Aspirin did not alter phospholipase activity, and Willems et al. (1982) suggested that two pools of CO exist: one as an exoenzyme and another located on the endoplasmic reticulum. The low levels of aspirin continuously fed to chicks in our experiment may only inhibit PGI2 production temporarily; hence, 6KPGF levels would not be depressed when measured in blood at 8 days. The data on fatty acids in liver tissues of biotin-deficient chicks were similar to the findings of Pearson et al. (1976), which showed elevated levelsof 16:1, 18:lw9, and 18:2a)6and decreased 20:4w6 at 15 days. At 15 and 20 days, 18:2co6 was elevated and 20:3w6 was decreased. Elevated 16:1 in liver during FLKS and biotin

deficiency in chickens was reported by several investigators (Roland and Edwards, 1971; Johnson et al, 1972; Whitehead et al., 1975). The reduction in liver 20:3w6 during biotin deficiency in chicks has not been reported; however, rats showed a significant reduction when fed a biotin-deficient diet (Kramer et al., 1984). In heart tissues of biotin-deficient chicks, lowered 20:3co6, and elevated 16:1 were observed at 15 and 20 days. The data indicate an impaired conversion of 18:2w6 to the PG fatty acid precursor 20:3w6 during biotin deficiency in chick liver at 15 and 20 days of age. The decrease of PG precursor substrates could reduce synthesis of PGs. Heart PGE2 levels were decreased during severe biotin deficiency. The decrease in PGE2 may contribute to ADS mortality in broilers as this PG is an important vasodilator of blood vessels (Claeys et a/.,1981a,b). Elevated 18:lw9 in liver of lipids of biotindeficient chicks could impair the desaturation of 18:2w6 and subsequent conversion to 20:4w6 (Naughton, 1981). The delta-6 desaturase enzyme is rate-regulating in the conversion of 18:2co6 to 18:3w6 and both 18:3co3 and 18:lw9 compete with 18:2w6 for this enzyme (Brenner and Peluffo, 1966; Mead, 1966; Naughton, 1981; Brenner, 1982). In the conversion of 18:3w6 to 20:3w6, malonyl-CoA is required for elongation and the enzyme providing the substrate carbon is biotin dependent (acetyl-CoA

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0 100 400 500 0(SM)

6-keto-PGF,-a

BIOTIN EFFECTS ON POLYUNSATURATED FATTY ACIDS C Tl8:2u6

m^

18:3u6

••^l20:3u6

A6

E

M^

20:4u6

A5

A , A * Desaturase eniymes E

» Elongation enzyme, (biotin dependent)

C

« Competition by elevated 18:3u3 and 18:lu9

carboxylase). Conceivably, biotin deficiency in the chick could impair the conversion of 18:2w6 to 20:4co6 at two steps (Figure 1). First, the elevated fatty acids, 18:3w3 and 18:lw9, could compete with 18:2w6 for the delta-6 desaturase enzyme and would reduce desaturation of 18:2co6. Second, a biotin deficiency may reduce malonyl-CoA and effectively lower 20:3w6 and 20:4a)6. In our experiments, liver 20:3w6 was reduced whereas 18:2w6 was elevated and in some cases 20:4M6 reduced and 18:lw9 increased. This indicates lowered desaturation of 18:2w6. Unfortunately 18:3w6 could not be detected with the present gas-liquid chromatography method. Therefore the elongation of this fatty acid could not be evaluated. The relationship of biotin to ADS in young chickens and to SIDS in human infants is supported by the fact that there is reduced liver biotin in both of these conditions. Liver and heart lipids from biotin-deficient chicks contained less 20:3w6 compared to biotin-adequate chicks. The decrease in 20:3w6 was also reported in SIDS infants. The results of this study show that PG precursors are reduced in biotin deficiency. Abnormal PG synthesis may be a mechanism by which biotin may influence the chick condition (lack of adequate prostaglandin vasodilator). The data from this work would indicate that biotin deficiency does impair the conversion of 18:2w6 to 20:4w6. REFERENCES Armstrong, J. M., N. Lattimer, S. Moncada, and J. R. Vane, 1978. Comparison of the vaso-depressor effects of prostacyclin and 6-oxo-prostaglandin F, with those of prostaglandin E2 in rats and rabbits. Br. J. Pharmacol. 62:125-130. Bannister, D. W., 1976. The biochemistry of fatty liver and kidney syndrome. Biochem. J. 156:167-173. Bannister, D. W., A. J. Evans, and C. C. Whitehead, 1975. Evidence for a lesion in carbohydrate metabolism in

fatty liver and kidney syndrome in chicks. Res. Vet. Sci. 18:149-156. Bligh, E. G., and W. J. Dyer, 1959. A rapid method of total lipid extraction and purification. Can J. Biochem. Physiol. 37:911-917. Brenner, R. R., 1974. The oxidative desaturation of unsaturated fatty acids in animals. Mol.Cell. Biol. 3:41-52. Brenner, R. R., 1982. Nutritional and hormonal factors influencing desaturation of essential fatty acids. Prog. Lipid Res. 20:41^17. Brenner, R. R., and R. O. Peluffo, 1966. Effect of saturated and unsaturated fatty acids on the desaturation in vitro of palmitic, stearic, oleic, linoleic and linolenic acids. J. Biol. Chem. 241:5213-5219. Bult, H., E. Wechsung, A. Houvenaghel, and A. G. Herman, 1981. Prostanoids and hemostasis in chickens: Anti- aggregating activity of prostaglandins E, and E 2 , but not of prostacyclin and prostaglandin D2. Prostaglandins 21:1045-1058. Claeys, M., E. Wechsung, A. G. Herman, and D. H. Nugteren, 1981a. Prostaglandin E2 is the prevalent metabolite of arachidonic acid formed by aortic tissue of the chicken. Arch. Int. Pharmacodyn. Ther. 249(2):312315. Claeys, M., E. Wechsung, A. G. Herman, and D. H. Nugteren, 1981b. Lack of prostacyclin biosynthesis by aortic tissue of the chicken. Prostaglandins 21:739-749. Fogerty, A. C , G. L. Ford, M. E. Willcox, and S. L. Clancy, 1984. Liver fatty acids and the sudden infant death syndrome. Am. J. Clin. Nutr. 39:201-208. Folch, J., M. Lees, and G.H.S. Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Granstrom, E., 1979. Sources of error in prostaglandin and thromboxane radioimmunoassay. Pages 229-238 in: Radioimmunoassay of drugs and hormones in cardiovascular medicine. M. Albertini, M. Da Prada, and B. A. Peskar, ed. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands. Heard, G. S.,R. L. Hood, and A. R.Johnson, 1983. Hepatic biotin and the sudden infant death syndrome. Med. J. Aust. 2:305-306. Hemsley, L. A., 1965. The "Fatty Liver and Kidney Syndrome" of young chickens. Vet. Rec. 77:124-126. Hertlendy, F., andH. V. Biellier, 1978. Prostaglandin levels in avian blood and reproductive organs. Biol. Reprod. 18:204-211. Hillier, K., and S. R. Dilley, 1974. Separation and radioimmunoassay of F 2 prostaglandins using silica gel micro columns. Prostaglandins 5:137-150. Hood, R. L., 1975. A radiochemical assay for biotin in biological materials. J. Sci. Food. Agric. 26:18471852. Hood, R. L., 1977. The use of linear regression analysis in the isotope dilution assay of biotin. Anal. Biochem. 79:635-638. Hood, R. L., A. R. Johnson, A. C. Fogerty, and J. A. Pearson, 1976. Fatty liver and kidney syndrome in chicks. II. Biochemical role of biotin. Aust. J. Biol. Sci. 29:429-441. Hulan, H. W., F. G. Proudfoot, and K. B. McRae, 1980. Effect of vitamins on the incidence of mortality and acute death syndrome ("Flip-Over") in broiler chickens. Poultry Sci. 59:927-931. Johnson, A. R., R. L. Hood, andJ. L. Emery, 1980. Biotin and the sudden infant death syndrome. Nature (Lond.)

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FIGURE 1. Effects of biotin deficiency on linoleate metabolism in chick liver.

1827

1828

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