G. Ananda Rao

Importance of Fetal and Neonatal Iron: Adequacy for Normal Development of Central Nervous System

Iron is an integral component or an essential cofactor of several enzymes such as aconitase, catalase, cytochrome C, cytochrome C reductase, cytochrome C oxidase, formiminotransferase, monoamine oxidase, myeloperoxidase, peroxidase, ribonucleotidyl reductase, succinic dehydrogenase, tyrosine hydroxylase, tryptophan pyrrolase and xanthine oxidase [1]. These enzymes are involved in a number of important pathways such as DNA synthesis, mitochondrial electron transport, catecholamine metabolism, neurotransmitter levels, and detoxification [1]. Iron is also involved in lipid metabolism. The multienzyme complex which catalyses the desaturation of stearoyl-CoA to yield the monounsaturated oleoyl-Coa (Δ 9-desaturase) contains not only cytochrome b5 but also a terminal desaturase enzyme which is a non-heure iron protein [2–4]. Thus, each desaturase enzyme complex contains two atoms of iron. Iron is also required for the production of polyunsaturated fatty acids [5]. Carnitine is necessary for the transport of long-chain fatty acids into the mitochondria for beta-oxidation. Its synthesis from trimethyl lysine involves two hydroxylases which require ferrous iron [6,7]. Hepatic levels of carnitine have been reported to be reduced in irondeficient rat pups compared to iron-supplemented controls [8]. Thus iron has an important role in various metabolic events related to lipids, such as oxidative degradation of fatty acids, synthesis of mono- and polyunsaturated fatty acids, plasmalogens and prostaglandins [9]. Although lipids play a key role in many health-related problems such as obesity, cancer and heart disease, investigations on the effects of iron deficiency on tissue lipid metabolism have been sparse.

Dietary arachidonic acid reduces fatty liver, increases diet consumption and weight gain in ethanol-fed rats

We fed young male Sprague-Dawley rats for 4 wk ad libitum liquid diets containing 34% of the calories as ethanol and 35% as fat with (AA+) and without (AA−) arachidonic acid (20∶4). Additional rats in the control groups were fed similar diets made isocaloric with dextrose with (CA+) and without (CA−) 20∶4. The liver triglyceride (TG) content of rats in the AA+ group was reduced ca. 3-fold over that of rats in the AA-group. The diet consumption and body wts of rats in the AA+ group were significantly greater than those of rats fed alcohol without the 20∶4 supplement (AA−). Also livers from rats in the AA+ group were as large as those from rats in control groups (CA+, CA−) and ca. twice as large as those from rats in the AA-group. The fatty acid composition of liver TG in rats fed the alcohol diet was similar to that of dietary fat. Levels of 20∶4 and docosatetraenoic acid (22∶4) in liver TG fatty acids from rats fed diets without arachidonate (AA−, CA−) were low (trace to 1.6%). After ingestion of arachidonic acid, 20∶4 increased to ca. 10% and 22∶4 to ca. 5%. The content of liver phospholipids was higher in livers of rats fed ethanol (AA−) than in those of controls (CA−).

Diet-induced alterations in the discoid shape and phospholipid fatty acid compositions of rat erythrocytes

For eight weeks young male rats were fed diets rich in 18∶2 (stock diet, or 10% corn oil, CO) or those devoid of 18∶2 (fat free, FF, or 10% hydrogenated coconut oil, HCNO). The CO and HCNO diets were fed in the absence or presence of eicosa-5,8,11,14-tetraynoic acid (TYA). When 18∶2 was excluded, an increase in the level of 16∶1, 18∶1 and 20∶3 and a decrease in 18∶2 was observed in the fatty acids of red cells. On feeding TYA, an increase in 18∶2 and in the case of the HCNO+TYA diet, a decrease of 12∶0 and 14∶0 was also observed. In all cases the levels of 20∶4 in erythrocyte fatty acids were similar. Saturated fatty acids were predominant in phosphatidyl choline (PC), lysophosphatidylcholine, (LPC) and sphingomyelin whereas unsaturated acids were predominant in phosphatidyl ethanolamine (PE), (PS), and phosphatidyl inositol (PI). Acids containing three or more double bonds comprised about 90% of the total acids in PI. In all the phospholipids, the characteristic changes in the composition of fatty acids were observed due to the exclusion of 18∶2 from the diet. However, changes due to the feeding of TYA were found only in PC and LPC. In rats fed the 18∶2-rich diet, about 60% of the red cells were discocytes. In those fed the 18∶2-free diet, the level of discocytes decreased to about 23%, and the levels of echinocytes II and III increased. The exclusion of 18∶2 for even a few days decreased the proportion of discocytes. The loss of discoid shape was reversed in a few days by feeding an 18∶2-rich diet. Fatty acid analysis of erythrocytes of rats of the various dietary manipulations showed that the change in the proportion of discocytes followed the change in the level of 18∶2.

Complexities in lipid quantitation using thin layer chromatography for separation and flame lonization for detection

The use of thin layer chromatography (TLC) for separation (using silica gel coated quartz rods) and subsequent flame ionization for detection (FID) was examined to determine whether this method could be used for the quantitation of lipids. However, response factors (RF) for various lipids were different and depended upon several variables including the amount of material analyzed. For example, RF were 3-fold greater when 10 μg of tripalmitin was analyzed as compared to 1 μg of the same material. The amount of lipid detected by FID was also dependent upon the rate at which it passed through the flame. During analysis of methylpentadecanoate, detector response increased with scan speed, while at all speeds it was completely removed from the rod. On the other hand, depending upon the amount of cholesterol or phospholipid analyzed, the response either increased, remained unchanged or decreased with scan speed. During a fast scan, detector response was reduced because some material remained on the rod. Thus, the detector response is influenced by sample volatility. In conclusion, there appears to be a complex relationship between detector response and the amount of heat available per microgram of sample. Since we could not find a direct correlation between detector response and sample quantity, it would be difficult to use TLC-FID as a tool for quantitating the components of a lipid mixture.

Reduction of hepatic stearoyl-CoA desaturase activity in rats fed iron-deficient diets.

The effect of feeding iron-deficient diets to rats on the hepatic stearoyl-CoA desaturase activity was examined since iron is present in the Δ9 desaturation system. Separate groups of rats were fed low iron diets without fat (FF-Fe) or containing either 14% hydrogenated coconut oil (HCNO-Fe) or 14% corn oil (CO-Fe) for 10 weeks. Diets supplemented with iron (FF+Fe, HCNO+Fe and CO+Fe) were fed to the corresponding control groups. Stearoyl-CoA desaturase activity in the liver microsomes of rats in the CO+Fe group (2.55±0.17 nmol oleate produced/min/mg protein) was about half of that in the HCNO+Fe (4.76±0.15) and FF+Fe (5.38±0.18) diet groups. In rats which were fed iron-deficient diets, hepatic desaturase levels were reduced significantly as compared to those of controls (1.0±0.06, CO-Fe; 2.11±0.13, HCNO-Fe; 3.65±0.1, FF-Fe). The hemoglobin (Hb) and hematocrit (Hct) levels in blood showed moderate iron depletion only in the CO-Fe group. Hence, dietary polyunsaturated fat promotes the onset of iron deficiency. Furthermore, even before the blood Hb and Hct values express iron depletion, the effect of feeding low iron diets was observed by the reduction of hepatic Δ9 desaturase activity in rats fed HCNO-Fe and FF-Fe diets.

Severe fatty liver in rats fed a fat-free ethanol diet, and its prevention by small amounts of dietary arachidonate

Rats were fed ethanol and a fat-free diet for 30 days to determine whether dietary fat is needed for the development of fatty liver. The severity of fatty liver was similar to that of rats fed an isocaloric diet with 35% fat. Small amounts (29 mg/day) of dietary arachidonic acid prevented alcoholic fatty liver. Rats fed either the alcohol (AF) or control (CF) fat-free diets developed essential fatty acid deficiency (EFAD) as measured by the triene/tetraene ratio of liver and plasma lipids. Rats fed arachidonic acid (AA, alcohol and CA, control diets) did not develop EFAD. Although EFAD alone did not cause the development of fatty liver, the combination of dietary ethanol and EFAD did. The ratios of 16∶1/16∶0 and 18∶1/18∶0 in liver lipids indicated that desaturase enzymes were less active and lipogenesis was reduced in rats fed the AA diet compared to those fed the AF diet. In contrast, stimulated lipogenesis appears to have been the cause of fatty liver in rats fed the AF diet.

Inadequate intake by growing rats of essential nutrients from liquid diets used for chronic alcohol consumption

Abstract In most studies on the biological effects of chronic alcohol ingestion, young rats were fed liquid diets containing 36% calories as ethanol. These diets were considered nutritionally adequate. However, the daily weight gain of alcohol-fed rats is small (2–4 g) as compared to that in rats maintained on an AIN-76 or NIH-07 diet (7 g). Alcohol-fed rats consume not only less calories but also less calcium, copper, iron, manganese, phosphorus and vitamin B 6 than those required for normal growth. A possible deficiency in the intake of other essential nutrients may also exist during chronic alcohol consumption.

Lieber-decarli alcohol diet modification to enhance growth in young rats

Abstract When male Sprague Dawley rats weighing ca 160 g were fed theLieber-DeCarli alcohol diet for 3 weeks, their growth rate was 3.8± 0.6 g/day. When the diet was supplemented with calcium phosphate (2.92 g/l), manangese carbonate (20.4 mg/l), ferric citrate (35 mg/l), zinc carbonate (9.3 mg/l), copper carbonate (1.75 mg/l), choline bitartrate (353 mg/l) and pyridoxine hydrochloride (1.2 mg/l), growth increased significantly (4.8±0.8 g/day). The daily weight gain was even greater when rats were fed the Lieber-DeCarli diet having instead of 36%, only 26% calories as alcohol (6.9±1.4 g/day). The amount of diet consumed per day also increased ca 50% as compared to the rats fed the 36% alcohol diet. Thus, the ingestion of more calories and minerals than that by rats fed the Lieber-DeCarli alcohol diet promoted a higher growth rate. Hence, supplementation of the 26% alcohol diet did not exhibit any marked effect on growth. Regardless of feeding the Lieber-DeCarli alcohol diet or its modifications, rats consumed similar amounts of alcohol (22–24 calories) per day. Our observations show that new investigations can now be carried out to examine the effect of chronic alcohol administration under conditions of improved growth in young rats.

Reduction of essential fatty acid deficiency in rats fed a low iron fat free diet.

Young male rats were fed ad libitum for 8 weeks a low iron fat-free (FF-Fe) diet or a fat-free diet supplemented with iron (FF+Fe). The relative levels of 16∶1 to 16∶0 and 18∶1 to 18∶0 in the total fatty acids of liver and other tissues (plasma, erythrocytes and intestinal mucosa) were considerably decreased because of a lack of dietary iron. In rats fed the FF-Fe diet, the levels of essential fatty acids (18∶2ω6+20∶4ω6) in tissues were 2-to 3-fold greater than in the corresponding tissues of rats fed the FF+Fe diet. Eicosatrienoic acid (20∶3ω9) levels in tissue lipids from rats fed the FF+Fe diet were high (8–16%), whereas they were low (2–5%) in the case of animals fed the FF-Fe diet. The proportion of 20∶4 in total fatty acids of tissues was 2-to 3-fold greater in rats fed the FF-Fe diet than when they were fed the FF+Fe diet. Therefore, the relative levels of 20∶3ω9/20∶4ω6 varied from 1-2.9 in tissue lipids of rats fed the FF+Fe diet, while it varied only from 0.2–0.3 in animals fed the FF-Fe diet. These results suggest that a lack of dietary iron may reduce the synthesis of 16∶1, 18∶1, 20∶3 and 20∶4 and the metabolism of 20∶4.

Dietary carbohydrate stimulates alcohol diet ingestion, promotes growth and prevents fatty liver in rats

Abstract When young male Sprague Dawley rats were fed ad libitum for 4 weeks a Lieber-DeCarli diet containing 36% of the total calories as ethanol, they consumed ca 50 ml of diet per day, gained 3–3.7 g/day and their livers contained ca 60 mg triglyceride/g. On the other hand, when rats were maintained on isocaloric diets containing 30%, 26% or 20% of calories as alcohol, the daily diet consumption (64, 67 and 88 ml respectively) and growth rate (4.2, 5.8 and 6.9 g respectively) increased markedly and their livers contained only 20–25 mg triglyceride/g. The amount of alcohol calories consumed per day or the alcohol consumption per 100 g body weight were comparable in the various diet groups. Since the reduction in alcohol calories was compensated for by including isocaloric amounts of maltose-dextrins, it is likely that the increased carbohydrate content stimulated diet ingestion, promoted growth and prevented the development of fatty liver despite chronic alcohol consumption.