Long-chain polyunsaturated fatty acids (LC-PUFA) during early development: contribution of milk LC-PUFA to accretion rates varies among organs.

Long-chain polyunsaturated fatty acids (LC-PUFA) accretion (essential for growth and neural development) was studied from late fetal throughout weaning age in the ferret, a species with maternal LC-PUFA sufficiency during pregnancy and lactation. The data show that a) accretion rate of LC-PUFA is rapid during early postnatal development, b) milk LC-PUFA decrease during lactation, c) adipose tissue LC-PUFA level is directly related to milk LC-PUFA level, while accretion in brain and liver exceeds dietary intake, d) accretion of arachidonic acid occurs earlier than docosahexaenoic acid, suggesting earlier development of n6-fatty acid endogenous synthesis.

Gastric proteolysis in preterm infants fed mother's milk or formula.

Gastric proteolysis is assumed to be low in the newborn (Britton & Koldovsky 1989). Postprandial pepsin output is significantly lower in preterm infants than adults, 589 vs. 3352U/kg, respectively (Armand et al. 1995, 1996). We now report on gastric proteolysis in preterm infants (gestation age, 29 weeks; postnatal age, 5-6 weeks) gavage-fed mother's milk or preemie formula. The data show that a) the nonprotein component is higher in human milk than formula, b) net proteolysis amounts to 15% of protein, c) gastric proteolysis is lower than lipolysis and, contrary to the latter, is not enhanced by milk feeding (Armand et al. 1996). We suggest that stomach pH, enzyme output, and food structure are the reasons for differences in gastric digestion of protein and fat in infants.

Protective function of human milk: the milk fat globule.

Human milk contains many components that protect the newborn against infection at a time when the infant's own defense mechanisms are poorly developed. Fat is one of the major nutrients in human milk. The fat is contained within milk fat globules composed of a core of triglyceride and a membrane consisting of phospholipids, cholesterol, proteins, and glycoproteins. Both the membrane and the core components can provide protection against microorganisms. The major protective membrane glycoproteins, mucin, and lactadherin are resistant to conditions in the newborn's stomach and maintain their structure and function even at low pH and in the presence of the proteolytic enzyme pepsin. The core triglycerides upon hydrolysis by digestive lipases (especially gastric lipase, which is well developed in the newborn) produce free fatty acids and monoglycerides, amphiphylic substances able to lyse enveloped viruses, bacteria, and protozoa. Therefore, in addition to its nutritional value, the fat in human milk has a major protective function.

Milk fat globule glycoproteins in human milk and in gastric aspirates of mother's milk-fed preterm infants.

Human milk fat globule (HMFG) glycoproteins can prevent infections by microorganisms in breast-fed infants; the MUC-1 mucin inhibits binding of S-fimbriated Escherichia coli to buccal mucosa, and lactadherin may prevent symptomatic rotavirus infections. In this study, the survival of these HMFG glycoproteins in the stomach of human milk-fed preterm infants (gestational age = 27.5 +/- 0.4 wk) was assessed, and levels in their mothers' milk determined, using specific RIAs. Butyrophilin, a major component of HMFG membrane that has no demonstrated antimicrobial activity, was studied for comparison. The levels of mucin, lactadherin, and butyrophilin in 41 milk samples of 20 mothers were 729 +/- 75, 93 +/- 10, and 41 +/- 3 microg/mL, respectively. Mucin and lactadherin were significantly higher in early milk samples (<15 d postpartum) than in later milk samples (15-90 d postpartum), whereas butyrophilin showed no such difference. Significant amounts of mucin and lactadherin were found in almost all gastric aspirates of human milk-fed infants, even 4 h after feeding (mucin, 270 +/- 30 microg/mL; lactadherin, 23.2 +/- 4.4 microg/mL), whereas butyrophilin was rapidly degraded in the majority of aspirates. Western blot analysis demonstrated that the immunoreactive mucin, lactadherin, and butyrophilin in the milk-fed gastric aspirates had the expected native molecular weights. Mucin and lactadherin survived at all gastric pH values, whereas butyrophilin was found only at pH > 4. Neither lactadherin nor butyrophilin were detected in gastric aspirates of formula-fed infants (gestational age = 27.8 +/- 0.5 wk), whereas the very low level of mucin (9.1 +/- 1.1 microg/mL) in this group is presumably cross-reacting gastric mucin. These results demonstrate that two HMFG glycoproteins implicated in prevention of infection, MUC-1 mucin and lactadherin, survive and maintain their integrity in the stomachs of human milk-fed preterm infants.

Molecular cloning of the bile salt-dependent lipase of ferret lactating mammary gland: an overview of functional residues.

Ferret lactating mammary gland bile salt-dependent lipase (BSDL, EC 3.1.1.-) has been cloned by RT-PCR. The open reading frame consists of 1869 nucleotides which encode 623 amino acids of the functional enzyme. When compared to other species, the greatest homology is observed between residues 1 and 484, with little or no homology at the C-terminal end where seven repeated segments of similar sequence are located. Ferret mammary gland BSDL retains residues involved in the active site and the tentative heparin binding site at similar positions in comparison to other milk or pancreatic BSDL. Other important items, such as binding peptide to chaperone molecular, phosphorylation site(s) or bile salt binding sites, were also tentatively located in well conserved regions of seven available BSDL sequences.

Gastric lipase and pepsin activities in the developing ferret: nonparallel development of the two gastric digestive enzymes.

BACKGROUND: Gastric lipase has an important compensatory function in neonatal fat digestion. The activity level of pepsin and its role in protein digestion is less well understood. We have, therefore, studied the ontogeny of lipase and pepsin in the ferret, a species with a neonatal fat digestion pattern similar to that of humans. METHODS: Gastric lipase and pepsin activities were quantified from the late fetal period throughout lactation, and were compared with those of the adult. RESULTS: The data show earlier ontogeny and much more rapid rise of lipase activity than of pepsin. Lipase activity was present during the last week of fetal development, whereas pepsin was detected only postnatally. Lipase activity was 72.8% +/- 14.2% and 153% +/- 9.95% and pepsin activity was 11.6% +/- 1.3% and 30.1% +/- 1.3% of the adult level at 2 and 4 wk of age, respectively. CONCLUSIONS: We conclude that lipase activity develops early and exceeds adult activity during the suckling period, when fat intake is very high. The low pepsin activity and high postprandial pH probably limit gastric proteolysis, thereby contributing to the structural and functional stability of milk proteins, many with protective or bioactive function in the gastrointestinal tract of the newborn.

Digestive enzymes in human milk: stability at suboptimal storage temperatures.

BACKGROUND: Women who return to work outside of the home while still breastfeeding must often store the expressed milk at less than optimal temperatures. Human milk provides digestive enzymes (amylase and lipase) that compensate in the newborn for immature pancreatic function. METHODS: We have assessed the stability of amylase and bile salt-dependent lipase after storage for 1-24 h at 15, 25, and 38 degrees C. RESULTS: Both enzymes were stable at 15 and 25 degrees C for 24 h, whereas at 38 degrees C there was a 15 and 20% decrease in lipase and amylase activity, respectively. The stability of milk lipoprotein lipase was also tested. This very labile enzyme was more stable in milk than previously reported for blood and tissues, i.e., 20 and 50% decrease in activity after storage at 15 or 25 degrees C for 24 h, respectively. A two-unit drop in milk pH by 24 h of storage would not affect the activity of digestive enzymes, which are stable at pH > 3.5. CONCLUSIONS: We conclude that milk provides the same compensatory digestive activity after short-term storage, even at relatively high temperature, as when fed fresh to the infant.

Effect of human milk or formula on gastric function and fat digestion in the premature infant.

The effect of diet, human milk or formula, on gastric function (lipase and pepsin activity, pH, and volume) and intragastric digestion of fat was assessed in 28 appropriate for gestational age preterm infants (gestational age, 28.9 +/- 1.4, 29.1 +/- 0.9, 29.5 +/- 0.6 wk; birth weight, 1.00 +/- 0.14 to 1.18 +/- 0.07 kg). The infants were fed either human milk (n = 11), SMA Super Preemie formula (n = 9), or Similac, Special Care formula (n = 8). Fasting and postprandial activity of digestive enzymes, pH, and gastric volume (measured before or during 50 min after gavage feeding) did not differ as a function of diet among the three groups of infants. Gastric lipase output, 23.1 +/- 5.1, 28.3 +/- 6.6, and 22.5 +/- 6.4 (U/kg of body weight) in human milk-, SMA SP-, or Similac SC-fed infants was comparable to the gastric lipase output of healthy adults fed a high fat diet (22.6 +/- 3.0). Pepsin output was, however, significantly lower (597 +/- 77, 743 +/- 97, and 639 +/- 142 U/kg of body weight) in human milk-, SMA SP-, and Similac SC-fed infants) than in healthy adults (3352 +/- 753 U/kg). The hydrolysis of dietary fat was 1.7-2.5-fold higher (p < 0.01) in human milk-fed infants than in infants fed either formula. We conclude that differences in type of feeding, i.e. different fatty acid profiles (long chain or medium chain triglycerides), different emulsions (natural or artificial), and different fat particle sizes do not affect the level of activity of gastric enzymes. However, the triglyceride within milk fat globules appears to be more accessible to gastric lipase than that within formula fat particles. We suggest that the contribution of gastric lipase to overall fat digestion might be greater in the newborn (a period of pancreatic insufficiency) than in the adult.

Digestive lipases of the newborn ferret: compensatory role of milk bile salt-dependent lipase.

The amount of mRNA hybridizing to bile salt-dependent lipase and to colipase-dependent lipase probes as well as their translation into active proteins were quantified in the adult and newborn pancreas and lactating mammary gland from the ferret, a species whose milk, similar to that of the human, has bile salt-dependent lipase. The concentration of colipase-dependent lipase mRNA correlated with the amount of activity found in the adult and newborn pancreas, whereas neither mRNA nor activity of this enzyme was detected in the kit pancreas or in the lactating mammary gland. These data indicate that colipase-dependent lipase is actually expressed in adult pancreas and might represent the main lipolytic system in the adult. mRNA hybridizing to the bile salt-dependent lipase probe used in this study were detected in adult and in newborn ferret pancreas as well as in lactating mammary gland. However, the bile salt-dependent lipase activity expressed in the newborn pancreas was very low when compared with the activity expressed either in the mammary gland or in the adult pancreas. These data argue for a compensatory role of milk bile salt-dependent lipase in lipid digestion in the newborn. The hydrolysis of dietary fat might be initiated by preduodenal lipase, the activity of which is only two times lower in the gastric mucosa of the newborn than in the adult ferret. The high concentration of mRNA hybridizing to the bile salt-dependent lipase probe associated with a very poor bile salt-dependent lipase activity and protein suggests either that these mRNA are very unstable or that they are poorly translated into an active pancreatic bile salt-dependent lipase.

Breastfeeding and the working mother: effect of time and temperature of short-term storage on proteolysis, lipolysis, and bacterial growth in milk.

BACKGROUND: Women who breastfeed have to store expressed milk while at work for later feeding to their infants; however, storage conditions are often not optimal. OBJECTIVE: Top assess microbial growth and stability of milk protein and lipid at 15 degrees C to 38 degrees C for up to 24 hours. METHODS: Sixteen healthy women who breastfed exclusively, either at home (n=11) or who expressed milk for their infants (n=5), were studied during early (1 month) or late (5 to 6 months) lactation. Expressed milk was stored at 15 degrees C, 25 degrees C, and 38 degrees C for 1 to 24 hours for quantitation of pH, proteolysis, and lipolysis; bacterial growth was quantified at 0, 4, 8, and 24 hours of storage. RESULTS: Milk pH decreased 2 units by 24 hours of storage at all temperatures tested. Proteolysis was minimal during milk storage at 15 degrees C or at 25 degrees C for 24 hours and was apparent only after 24 hours of storage at 38 degrees C. Lipolysis was rapid, starting in the first hours of storage and progressing to 8% at 24 hours. Thus, while the greatest increment in proteolysis products was a 40% increase above baseline after 24 hours of storage at 38 degrees C, free fatty acid concentration at this storage time was 440% to 710% higher than in freshly expressed milk. Bacterial growth was restricted mainly to nonpathogens, was minimal at 15 degrees C throughout the 24 hours of storage, was low at 25 degrees C for the first 4 to 8 hours, and was considerably higher at 38 degrees C even during the relatively short period of 4 hours. CONCLUSIONS: Storage of human milk is safe at 15 degrees C for 24 hours, whereas at 25 degrees C it is safe for 4 hours. Milk should not be stored at 38 degrees C. Minimal proteolysis during storage suggests that milk proteins probably maintain their structure and function during short-term storage, while the marked lipolysis might slow bacterial growth during this time.

Plasma lecithin: cholesterol acyltransferase and plasma lipolytic activity in preterm infants given total parenteral nutrition with 10% or 20% Intralipid.

The effect of 10% or 20% Intralipid on lipid clearing enzymes, plasma lipids and apoproteins was investigated during the first 5 days after birth in 37 premature infants maintained on total parenteral nutrition; 21 infants received 20% and 16 received 10% Intralipid, respectively. Lipid was infused over a 20-h period at rates of 1, 2 and 3 g/kg/day on consecutive days. Plasma lecithin: cholesterol acyltransferase (LCAT) activity was low and increased significantly (p<0.05) only during infusions of 3 g/kg/day in both groups of infants. Plasma lipolytic activity was generally not affected by the regimen or preparation (10% or 20%) of Intralipid infused, except for higher (p<0.05) levels at 3 g/kg/day of 20% compared with prelipid infusion. Plasma triglyceride concentrations wer similar after 10% or 20% Intralipid, whereas plasma total cholesterol was significantly higher during infusion of 2 and 3 g/kg/day of 10% compared with 20% Intralipid. The efficient clearing of 20% Intralipid might be related to the lower lecithin: triglyceride ration which is compatible with the low LCAT activity of premature infants.

Dietary fat modulates gastric lipase activity in healthy humans.

The aim of this study was to determine whether the amount of dietary fat modulates the activity of gastric lipase in humans. Gastric juice was collected from six healthy subjects after 2-wk periods of either a high-fat (50% of energy as fat) or low-fat (25% of energy as fat) diet. The collection period lasted 2 h, the first hour under baseline conditions and the second hour after pentagastrin stimulation (6 micrograms/kg body wt). Gastric lipase and pepsin activities were quantitated at 15-min intervals and total enzyme outputs were calculated. Under baseline conditions there was a tendency for higher output of gastric lipase and pepsin after the high-fat diet than after the low-fat diet (gastric lipase: 745 compared with 446 U/h, pepsin: 107,677 compared with 78,505 U/h). The difference in output between diet groups was significant after pentagastrin stimulation (gastric lipase: 1323 compared with 875 U/h, pepsin: 191,751 compared with 128,961 U/h, for high-fat compared with low-fat diet, respectively, P < 0.05). This study is the first to report that a high-fat diet leads to an increase in the activity of gastric enzymes in humans.

Cardiorespiratory effects produced by blockade of excitatory amino acid receptors in cats.

The aim of our study was to determine the role of excitatory amino acids in controlling cardiorespiratory activity. For this purpose we administered an antagonist of both N-methyl-D-aspartate (NMDA) and non-NMDA receptors (kynurenic acid), and an antagonist of the NMDA receptor complex (dizocilpine, more commonly known as MK-801) i.v. to chloralose-anesthetized cats while monitoring tracheal air flow, tidal volume, respiratory rate, inspiratory and expiratory durations, end tidal CO2, arterial blood pressure and heart rate. Administration of kynurenic acid in doses of 350 and 500 mg/kg produced respiratory depression as reflected by decreases in respiratory minute volume and increases in end tidal CO2. Inspiratory duration was increased with both doses and apnea (occurring during expiration) was observed with the high dose. Apnea was preceded by an apneustic pattern of breathing. Both doses resulted in an increase in blood pressure and, with the high dose, a later decrease in blood pressure was noted. Dizocilpine in doses ranging from 0.03 to 1 mg/kg produced dose-related decreases in respiratory minute volume, and increases in end tidal CO2. In addition, dizocilpine produced increases in inspiratory duration, an apneustic pattern of breathing and apnea (occurring during inspiration). Effects on blood pressure were similar to those observed with kynurenic acid. It is concluded that blockade of excitatory amino acid receptors results in pronounced effects on cardiorespiratory activity.

Human mammary gland function at the onset of lactation: medium-chain fatty acid synthesis.

The onset of medium-chain fatty acid synthesis in the human mammary gland was investigated. Colostrum and serum were collected from 31 healthy women and the fatty acid composition of total lipid was analyzed by gas-liquid chromatography. Although colostrum/serum ratios for most fatty acids range from 0.7-2.4, very low levels of 10:0 and 12:0 were present in serum lipids as compared to much higher concentrations of these fatty acids in colostrum lipids (colostrum/serum ratio 16.23 and 17.11 for 10:0 and 12:0, respectively). We have previously found that medium-chain fatty acid levels are very low in prepartum mammary secretions (6-10 wk before term delivery) but are higher and similar in colostrum of women who deliver preterm (3-14 wk) or at full term. The data indicate that parturition, irrespective of length of pregnancy, is the trigger for medium-chain fatty acid synthesis in the human mammary gland.

Fat absorption in premature infants: medium-chain triglycerides and long-chain triglycerides are absorbed from formula at similar rates.

Fat absorption from two different premature infant formulas and one full-term formula containing three different fat blends was investigated in two groups of premature infants. The first group of nine infants (gestational age, 29.1 +/- 0.88 weeks; postnatal age, 3.13 +/- 0.71 weeks) was fed alternately for 1 week each SMA preterm formula containing either high levels (50%) of medium-chain triglycerides (MCT) (6:0, 8:0, and 10:0) or high levels (86%) of long-chain triglycerides (LCT) (greater than or equal to C12). Except for fat blends, the formulas were otherwise identical. The second group of 11 infants (gestational age, 30.5 +/- 0.77 weeks, studied at a postnatal age of 4.33 +/- 0.91 weeks) was fed for 1 week a full-term infant formula, S-26, containing 98% LCT. Fat absorption (studied during a 3-day fat balance period) was similar irrespective of fat blend: 89.08 +/- 2.37% during feeding of preterm SMA, 50% MCT; 87.0 +/- 3.81% during feeding of preterm SMA, 86% LCT; and 83.00 +/- 2.89% during feeding of S-26, 98% LCT. Weight gain (grams per day) and increase in length (centimeters per day) were 23.2 +/- 1.7, 21.20 +/- 1.7, and 14.28 +/- 2.9, and 0.17 +/- 0.06, 0.16 +/- 0.04, and 0.22 +/- 0.07 during feeding of the three fat blends, respectively. Lipase activity levels in fasting gastric aspirates were higher during feeding of the LCT than the MCT formula. The possible stimulation of gastric lipase secretion secondary to long-chain fatty acid stimulation of cholecystokinin secretion might be related to the efficient digestion of formula fat, irrespective of triglyceride-fatty acid chain length.(ABSTRACT TRUNCATED AT 250 WORDS)

Lecithin:cholesterol acyltransferase (LCAT) activity during lipid infusion in premature infants.

Plasma cholesterol and lecithin concentrations are regulated by the serum enzyme lecithin: cholesterol acyltransferase (LCAT). LCAT activity is low in cord blood of premature infants, suggesting that in these infants the hypercholesterolemia associated with Intralipid infusion might be due to low LCAT activity. The serum LCAT activity has not been quantitated in preterm infants receiving intravenous fat emulsions. We have therefore quantitated LCAT activity in eleven premature infants maintained on total parenteral nutrition (TPN). Ten infants were studied during the first 2 weeks after birth; they received daily infusions of Intralipid at a rate of 0.5-2.0 g/kg/day over 15 h. One infant received 3.8 g/kg/day during the second week. In addition to LCAT, serum apoprotein A1 (the cofactor of LCAT), cholesterol, triglycerides, and free fatty acids were quantitated. Blood specimens were taken before the start of the infusion and 15-45 min before its completion. The LCAT activity and apoprotein A1 concentrations remained, respectively, 21-24% and 30-35% of adult levels. However, serum cholesterol levels remained in the normal range during the fat infusion. It remains to be established whether low LCAT activity and apoprotein A1 levels are due to the administration of Intralipid (which lowers LCAT activity in rats), to the lack of enteral feedings, or to prematurity per se. Our data suggest that administration of Intralipid at a rate not exceeding 1-2 g/kg/day does not impair the clearing of Intralipid-lecithin and the metabolism of cholesterol.

Lipase and pepsin activity in the gastric mucosa of infants, children, and adults.

The range of activity and the location of lipase and pepsin were determined in the stomach and duodenum of infants, children, and adults. The range of lipase activity in biopsy specimens from the gastric body, in 29 subjects aged from 3 months to 26 years, was 1.8-5.3 U/mg protein (1 U is 1 mumol [3H]oleic acid released from tri-[3H]olein per minute). There were no significant differences among age groups (5-19 months, 2-4 years, 6-10 years, 11-13 years, and 15-26 years). Lipase activity was low or undetectable in the gastric antrum of all subjects. Pepsin activity in specimens from the gastric body ranged from 180 to 780 pepsin units/mg protein (using hemoglobin as substrate). The antrum had significantly lower pepsin activity (P less than 0.001) than the gastric body. As with lipase activity, there were no statistically significant differences in pepsin activity among age groups. Lipase and pepsin activity was also quantified in pinch biopsy specimens from the duodenum and duodenal bulb in 13 subjects. Contrary to lipase activity, which was almost completely absent from the duodenum or duodenal bulb, these sites contained low pepsin activity (9-78 pepsin units/mg protein). The data show that in infants and children, as previously reported in adults, gastric lipase is localized primarily in the gastric body. Tissue pepsin levels and localization, reported here for the first time, are similar to those of lipase, although, contrary to lipase, the gastric antrum has considerable pepsin activity. The identical levels of lipase and pepsin activities in infants, children, and adults indicate that the gastric phase of nutrient digestion is well developed at birth.

Cocaine acts in the central nervous system to inhibit sympathetic neural activity.

Cocaine was administered i.v. to decerebrate cats while monitoring cardiac preganglionic sympathetic nerve activity (SNA), arterial blood pressure (BP) and heart rate (HR). Cocaine, 4 mg/kg i.v., reduced SNA by 55 +/- 6%, but did not significantly affect BP or HR. Cocaine, in doses that were ineffective by the i.v. route, was administered into the vertebral artery and produced decreases in SNA, BP and HR in anesthetized cats. Administration of cocaine into the carotid artery was without effect. Topical administration of cocaine to the intermediate area of the ventrolateral medullary surface (25 micrograms/side) evoked hypotension and bradycardia. Nisoxetine, an inhibitor of norepinephrine uptake, applied bilaterally to the intermediate area (30 micrograms/side) exerted a similar hypotensive effect. Lidocaine administered in doses equivalent to those of cocaine had no significant effect on SNA when given i.v. or on BP when given into the vertebral artery. These results indicate that cocaine inhibits central sympathetic outflow and that the site of action appears to be in the hindbrain at a site that is reached by placement of the drug at the intermediate area of the ventrolateral medulla. The data also indicate that the mechanism of action of cocaine to inhibit sympathetic outflow may be unrelated to its local anesthetic action and may involve inhibition of catecholamine uptake in the ventrolateral medulla.

Factors determining whether cocaine will potentiate the cardiac effects of neurally released norepinephrine.

The purpose of the present study was to re-evaluate the effects of cocaine on cardiac responses elicited by sympathetic nerve stimulation. Cats anesthetized with pentobarbital and subjected to spinal cord transection were used. Control heart rate increases were obtained to submaximal stimulation of postganglionic accelerator nerves, before and after i.v. bolus doses of cocaine ranging from 0.0625 to 2.0 mg/kg. Maximal potentiation of heart rate increases elicited by nerve stimulation were observed with 0.25 mg/kg. In precocaine controls, stimulation increased sinus rate by 31 +/- beats/min; 30 sec to 1 min after cocaine (0.25 mg/kg), stimulation increased sinus rate by 55 +/- beats/min. Maximal potentiation (80 +/- 10%) was observed at 30 sec to 1 min after cocaine administration, and was usually over by 45 to 60 min later. Cocaine was repeated twice (0.25 mg/kg i.v.) at hourly intervals and the magnitude of potentiation was only 19 +/- 6 and 24 +/- 4%, respectively, indicating that tachyphylaxis had developed toward cocaine's potentiating effect. Dose-response studies indicated that as little as 0.0625 mg/kg of cocaine can potentiate heart rate increases elicited by sympathetic nerve stimulation. Doubling the dose to 0.125 mg/kg, and again to 0.25 mg/kg, resulted in a linear dose-related increase in the magnitude of potentiation. However, doubling the dose again to 0.5 mg/kg and increasing this dose by 4-fold to 2 mg/kg did not result in additional potentiation. Indeed, there was a significant drop-off in the magnitude of potentiation to nerve stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)