Research Notes: Fatty Acid Metabolism

Omega oxidation

From http://www.mta.ca/~jstewart/BC2001/FA.html

The poorly soluble fatty acids are transported to other tissues bound to the soluble protein serum albumin (in many animals). The transport of the long chain fatty acids in the cell is accomplished by the fatty acid-binding proteins - again by binding the poorly soluble fatty acids to the interior of this small soluble protein.

Generally the liver is lipogenic (produces lipids) and the muscles and heart are the major sites of fatty acid oxidation.

Fatty acids produce much more energy than the oxidation of carbohydrates. One mole of palmitic acid (the most prevalent biological fatty acid) can produce the equivalent of 129 moles of ATP. Using the standard biological free energy of ATP hydrolysis, compared to the total energy release by combusion of palmitate to carbon dioxide we see that the efficiency is about 40%. Under biological conditions though (mainly through manipulation of concentrations) the efficiency can reach upwards of 80.

The oxidation of fatty acid occurs in the mitochondrial matrix and is referred to as beta-oxidation since the actual carbon being oxidize is beta to the carboxylate group (numbering from the carboxylate carbon, this would be carbon number 3)

Fatty acids are oxidized to two carbon units with the shortened chain going through the process again and again. Hence this could be referred to as a spiral pathway (in comparison to the linear glycolytic pathway and the cyclical citric acid cycle).

The two carbon unit is the now familiar acetyl-CoA.

Proof for the b-processing, or two carbon processing of fatty acids was first offered by Knoop in 1904. He fed dogs long chain fatty acids tagged with a benzene group on the omega carbon (the end carbon). He used even chained and odd chained fatty acids, reasoning that with the former the remainder would be phenylacetic acid while with the latter the product should be benzoic acid. Since both of these are detoxified in the liver by forming a peptide bond with glycine and then excreting the produce in urine, he collected dog urine and looked for the products. Obviously he found what we would expect or we wouldn't be talking about it!

There are four phases of oxidation of fatty acids

1. Activation of the fatty acid (reaction with ATP to adenylate the carboxylate group, FA-AMP plus pyrophosphate PPi). The pyrophosphate is rapidly hydrolyzed by pyrophosphatase to 2Pi. This diphosphoanhydride is also a 'high energy' bond and is the equivalent of an ATP. Thus we expend the equivalent of 2 ATP in the activation process. Finally the FA-adenylate is used to synthesize FA-CoA (an acyl-CoA) by the nucleophilic attach of the sulphur of CoA. Enzyme: fatty acyl-CoA ligase.

2. A trans-esterification of FA-CoA and L-carnitine to produce FA-Carnitine and free CoA--Enzyme: carnitine palmitoyltransferase I. It is the FA-carnitine that is transported into the mitochondrial matrix, not the CoA derivative. The main purpose of this reaction is to keep separate the pools of CoA in in the cytosol and in the mitochondrial matrix - recall that CoA is a control factor in a number of enzymes, most particularly pyruvate dehydrogenase of the mitochondria. Mixing the two pools of this cofactor would make compartmentalization of fatty acid oxidation (mitochondrial) and fatty acid synthesis (cytosolic) ineffective.

3. Transport into the mitochondria (a translocase that is an antiport - FA-Carnitine in, carnitine out). Inside the mitochondrial matrix a second enzyme, carnitine palmitoyltransferase II, resynthesizes the FA-CoA and liberates carnitine.

4. b-oxidation (four steps) occurs producing acetyl-CoA until the long chain fatty acid is completely degraded.

eg, Palmitoyl-CoA + 7 CoA + 7 FAD + 7 NAD+ + 7H2O --> 8 Acetyl-CoA + 7FADH2 + 7 NADH + 7H+

One way to keep account is n Carbons produces n/2 Acetyl CoA, n/2-1 each of FADH2 and NADH

The reactions of this spiral, just as we've seen before, incrementally changes the C/C sigma bond that is to be broken by

• introduction of a double bond (dehydrogenase)
• hydration of the double bond to an alcohol (OH on the b-carbon, this is chiral, L-3-hydroxy- )
• oxidation of the alcohol to a carbonyl group (dehydrogenase)
• nucelophilic attack of the S of a CoA at the carbonyl carbon (electron deficient) and cleavage of the bond between the a and the b carbon and, in the case of palmitate, production of myristoyl-CoA 14 carbons.

The acetyl-CoA produced can now be oxidized in the Citric Acid Cycle to carbon dioxide and reduced cofactors. The reduced cofactors of the Citric Acid Cycle are used to reduce the electron transport chain, and eventually, molecular oxygen as we have seen before.

Control of beta-Oxidation

Control of the process rests with carnitine palmitoyltransferase I that acts as a gatekeeper of entry into the mitochondrial. Once the carnitine ester is formed, the fate of that molecule is fairly well sealed - this is the committing step. FA-carnitine will be taken into the mitos and oxidized. The controlling compound, the allosteric effector of CPT I, is malonyl-CoA. This is the first compound on the road to SYNTHESIZING fatty acids in the mitochondria! In other words, the simultaneous synthesis and oxidation of fatty acids doesn't occur.

The source of the malonyl-CoA? It is made by the carboxylation of AcetylCoA by acetylCoA carboxylase (isoform 1 in the liver and isoform 2 in muscle and heart). The cytosolic acetylCoA used to synthesize fatty acids is made from excess citrate produced in the Citric Acid Cycle and transported to the cytosol. If FAs are being synthesized, malonyl-CoA levels are relatively high, and, by shutting down CPT I, fatty acids are not transported into the mitchondria.

Mol Genet Metab. 2007 Sep 5.
Choice of oils for essential fat supplements can enhance production of abnormal metabolites in fat oxidation disorders.
Roe CR, Roe DS, Wallace M, Garritson B.
Institute of Metabolic Disease, Baylor University Medical Center, 3812 Elm Street, Dallas, TX, USA.

Patients with mitochondrial long-chain fat oxidation deficiencies are usually treated with diets containing reduced fat and increased carbohydrate, at times via gastrostomy feeding. To ensure adequate intake of essential fatty acids, supplements are provided to their diets using commercially available oils. These oils contain large quantities of non-essential fats that are preferentially oxidized and produce disease-specific metabolites (acyl-CoA intermediates) due to the genetic defect. This study describes the concentrations of these intermediates as reflected by acylcarnitines as well as the % contribution from each of four fatty acids: palmitate, oleate, linoleate, and alpha-linolenate when incubated with fibroblasts from patients with VLCAD, LCHAD, and trifunctional protein (TFP) deficiencies. Palmitate and oleate produce the majority of disease-specific acylcarnitines with these defective cell lines (79-94%) whereas linoleate and linolenate produced less (6-21%). On average, the amount of acylcarnitines decreased with increasing unsaturation (C18:1>C18:2>C18:3:34%>11%>3%, respectively. This relationship may reflect the "gatekeeper" role of carnitine palmitoyltransferase I (CPT I). A diet comparison between Canola and a combination of Flax/Walnut oils revealed that the latter, containing the least amount of non-essential fats, reduced blood acylcarnitine levels by 33-36%. The etiology of the severe peripheral neuropathy of TFP deficiency may result from the unique metabolite, 3-keto-acyl-CoA, after conversion to a methylketone via spontaneous decarboxylation. Essential fatty acid supplementation with oils should consider these findings to decrease production of disease-specific acyl-CoA intermediates.

Mol Cell Endocrinol. 2007 Mar 15.
Altered lipid catabolism in the vitamin A deficient liver.
Kang HW, Bhimidi GR, Odom DP, Brun PJ, Fernandez ML, McGrane MM.
Departments of Nutritional Sciences and Molecular and Cellular Biology, The University of Connecticut, 3624 Horsebarn Hill Road Extension, Storrs, CT, USA.

The transcriptome pattern of metabolic genes in vitamin A deficient (VAD) liver has been compared to the vitamin A-sufficient (VAS) state using the Mouse 32k oligonucleotide (70mer) array. In VAD liver there was a decrease in expression of genes encoding enzymes of mitochondrial fatty acid (FA) oxidation; these genes included fatty acyl CoA ligase, carnitine o-palmitoyl transferase 1, medium chain acyl-CoA dehydrogenase, 3-ketoacyl CoA thiolase, and citrate synthase. Particularly affected was peroxisome metabolism, as genes encoding enzymes of peroxisomal FA oxidation and transport proteins were differentially expressed. These genes included those encoding acyl-CoA oxidase 1, the peroxisomal bifunctional enzyme, peroxisomal thiolase, and carnitine o-octanoyl transferase, the enzyme involved in shuttling FAs from the peroxisome to the mitochondrion. Most genes that were differentially expressed with chronic vitamin A depletion were responsive to treatment with all-trans retinoic acid (RA). Consistent with the decreased expression of genes involved in FA oxidation, we found an increase in hepatic macrocytic lipid accumulation and triglyceride levels. The relevant nuclear receptor gene that was differentially expressed in the VAD liver was that encoding the peroxisome proliferator-activated receptor (PPAR) alpha, the mRNA levels for which were decreased in VAD liver and increased with all-trans RA treatment. Down regulation of the PPAR alpha gene is the likely cause of the altered expression pattern of the above metabolic genes in VAD liver.

Am J Physiol Endocrinol Metab. 2007 Feb 20.
Skeletal muscle mitochondrial FAT/CD36 and palmitate oxidation are not decreased in obese women.
Holloway GP, Thrush AB, Heigenhauser GJ, Tandon NN, Dyck DJ, Bonen A, Spriet LL.
Human Health and Nutritional Sciences, University of Guelph, Guelph, Canada.

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean (BMI = 23.3 +/- 0.7 kg∙m(-2)) and obese women (BMI = 37.6 +/- 2.2 kg∙m(-2)). The mitochondrial marker enzymes, citrate synthase (-34%), β-hydroxyacyl-CoA dehydrogenase (-17%) and cytochrome c oxidase (-32%) were reduced (P<0.05) in obese participants, indicating mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate, however fatty acid oxidation was reduced at the whole muscle level by 28% (P<0.05) in the obese. Mitochondrial FAT/CD36 did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r=0.67, P<0.05). It is concluded that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.

Regul Pept. 2006 Nov 14.
Adiponectin modulates carnitine palmitoyltransferase-1 through AMPK signaling cascade in rat cardiomyocytes.
Li L, Wu L, Wang C, Liu L, Zhao Y.
Department of Physiology and Pathophysiology, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China.

Adiponectin, an adipocyte-derived polypeptide hormone, plays an important role in regulating fatty acid oxidation. beta-oxidation of fatty acids supplies most of the cardiac energy and carnitine palmitoyltransferase (CPT)-1 serves as a key regulator during this process. To characterize the potential effects of adiponectin on CPT-1, we incubated rat neonatal cardiomyocytes with globular adiponectin (gAd). Results showed that gAd promoted the activity and mRNA expression of CPT-1. The underlying signal pathway involved in this modulatory effect was further investigated. Inhibition of AMP-activated protein kinase (AMPK) with adenine 9-beta-d-arabinofuranoside (AraA) completely abrogated gAd-mediated AMPK and acetyl coenzyme A carboxylase (ACC) phosphorylation and suppressed the promotion of CPT-1 activity. gAd also induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and peroxisome proliferator-activated receptor (PPAR)-alpha, which was inhibited by AraA. SB202190, a p38MAPK inhibitor, blocked gAd-stimulated PPAR-alpha phosphorylation. When AMPK and/or p38MAPK was inhibited, gAd-enhanced mRNA expression of CPT-1 was partially reduced. In conclusion, our study suggests that the activation of AMPK signaling cascade participates in the promotion effect of gAd on CPT-1.

Am J Physiol Endocrinol Metab. 2006 Mar.
Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation.
Bezaire V, Bruce CR, Heigenhauser GJ, Tandon NN, Glatz JF, Luiken JJ, Bonen A, Spriet LL.
Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, ON, Canada.

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 muM) palmitate oxidation was decreased by 95% (P<0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P<0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.

Nat Med. 2003 Jun.
Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production.
Obici S, Feng Z, Arduini A, Conti R, Rossetti L.
Department of Medicine, Diabetes Research and Training Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, USA.

The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where the LCFAs undergo beta-oxidation. To investigate the mechanism(s) by which central metabolism of lipids can modulate energy balance, we selectively reduced lipid oxidation in the hypothalamus. We decreased the activity of CPT1 by administering to rats a ribozyme-containing plasmid designed specifically to decrease the expression of this enzyme or by infusing pharmacological inhibitors of its activity into the third cerebral ventricle. Either genetic or biochemical inhibition of hypothalamic CPT1 activity was sufficient to substantially diminish food intake and endogenous glucose production. These results indicated that changes in the rate of lipid oxidation in selective hypothalamic neurons signaled nutrient availability to the hypothalamus, which in turn modulated the exogenous and endogenous inputs of nutrients into the circulation.

Am J Physiol Endocrinol Metab. 2004 Oct.
Insulin stimulates fatty acid transport by regulating expression of FAT/CD36 but not FABPpm.
Chabowski A, Coort SL, Calles-Escandon J, Tandon NN, Glatz JF, Luiken JJ, Bonen A.
Department of Human Biology and Nutritional Sciences, University of Guelph, Canada.
[ Free full text ]

Because insulin has been shown to stimulate long-chain fatty acid (LCFA) esterification in skeletal muscle and cardiac myocytes, we investigated whether insulin increased the rate of LCFA transport by altering the expression and the subcellular distribution of the fatty acid transporters FAT/CD36 and FABPpm. In cardiac myocytes, insulin very rapidly increased the expression of FAT/CD36 protein in a time- and dose-dependent manner. During a 2-h period, insulin (10 nM) increased cardiac myocyte FAT/CD36 protein by 25% after 60 min and attained a maximum after 90-120 min (+40-50%). There was a dose-dependent relationship between insulin (10(-12) to 10(-7) M) and FAT/CD36 expression. The half-maximal increase in FAT/CD36 protein occurred at 0.5 x 10(-9) M insulin, and the maximal increase occurred at 10(-9) to 10(-8) M insulin (+40-50%). There were similar insulin-induced increments in FAT/CD36 protein in cardiac myocytes (+43%) and in Langendorff-perfused hearts (+32%). In contrast to FAT/CD36, insulin did not alter the expression of FABPpm protein in either cardiac myocytes or the perfused heart. By use of specific inhibitors of insulin-signaling pathways, it was shown that insulin-induced expression of FAT/CD36 occurred via the PI 3-kinase/Akt insulin-signaling pathway. Subcellular fractionation of cardiac myocytes revealed that insulin not only increased the expression of FAT/CD36, but this hormone also targeted some of the FAT/CD36 to the plasma membrane while concomitantly lowering the intracellular depot of FAT/CD36. At the functional level, the insulin-induced increase in FAT/CD36 protein resulted in an increased rate of palmitate transport into giant vesicles (+34%), which paralleled the increase in plasmalemmal FAT/CD36 (+29%). The present studies have shown that insulin regulates protein expression of FAT/CD36, but not FABPpm, via the PI 3-kinase/Akt insulin-signaling pathway.

J Child Neurol. 2002 Dec.
Role of carnitine and fatty acid oxidation and its defects in infantile epilepsy.
Tein I.
Division of Neurology, The Hospital for Sick Children, 555 University Ave., Toronto, ON.

Defects in fatty acid oxidation are a source of major morbidity and are potentially rapidly fatal. Fatty acid oxidation defects encompass a spectrum of clinical disorders, including recurrent hypoglycemic, hypoketotic encephalopathy or Reye-like syndrome in infancy with secondary seizures and potential developmental delay, progressive lipid storage myopathy, recurrent myoglobinuria, neuropathy, and progressive cardiomyopathy. As all of the known conditions are inherited as autosomal recessive diseases, there is often a family history of sudden infant death syndrome in siblings. Early recognition and prompt initiation of therapy and the institution of preventive measures may be life saving and significantly decrease long-term morbidity, particularly with respect to central nervous system sequelae. Seizures may be the result of cerebral bioenergetic failure associated with acute episodes of hypoglycemic, hypoketotic encephalopathy, or hypoxic-ischemic encephalopathy in the context of cardiac arrhythmias and/or cardiomyopathy. This review provides an overview of the fatty acid oxidation pathway and the central role of carnitine, as well as a discussion of normal fasting adaptation and the critical metabolic adaptations that occur at birth. The increased vulnerability of infants and young children to fasting and defective fatty acid oxidation is discussed in the context of the heightened bioenergetic demands of the developing brain. Clinical and laboratory features of specific genetic defects in fatty acid oxidation, approaches to diagnosis, and current treatment methodologies are described. Indications for carnitine supplementation in childhood epilepsy are also discussed.

J Am Diet Assoc. 2002 Dec.
Management of fatty acid oxidation disorders: a survey of current treatment strategies.
Solis JO, Singh RH.
Sarah Lawrence College, Bronxville, NY, USA.

Standardization of the nutritional care for patients with fatty-acid oxidation disorders is lacking. A literature review and national survey of metabolic dietitians describes the range of therapeutic strategies currently employed in the U.S. to treat patients with fatty-acid oxidation disorders. Questionnaire responses provided by dietitians specializing in metabolic disorders evaluated practices used for treatment of fatty acid oxidation disorders, medium-chain acyl-CoA dehydrogenase deficiency (MCAD), very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), long-chain acyl-CoA dehydrogenase deficiency (LCAD), and Trifunctional Protein deficiency (TFP). This survey reveals a significant lack of evidence supporting the protocols in use. Recent advances in tandem mass spectrometry technology promises an increase in the number of identified patients with fatty-acid oxidation disorders, which reinforces the need for comprehensive, clinical research studies to determine optimal care for patients with these genetic disorders.

Curr Opin Pediatr. 2000 Oct.
New developments in the pathophysiology, clinical spectrum, and diagnosis of disorders of fatty acid oxidation.
Treem WR.
Division of Pediatric Gastroenterology and Hepatology, Duke University Medical Center, Durham, North Carolina, USA.

Fatty acid oxidation disorders are among the most common inborn errors of metabolism affecting infants and children. Recognition of this family of defects is critical because careful dietary monitoring, avoidance of fasting, and prompt intervention during common childhood illness can prevent catastrophic cardiac and metabolic decompensation. This review focuses on new molecular and clinical diagnostic aspects of several of these disorders. Recent papers highlight the recognition that the clinical spectrum of disorders of fatty acid oxidation goes far beyond the stereotypical Reyes-like presentation or cardiomyopathy, and now encompasses more cases of sudden infant death syndrome, fulminant hepatic failure, and severe complications during pregnancy.

J Inherit Metab Dis. 1999 Jun.
Recognition and management of fatty acid oxidation defects: a series of 107 patients.
Saudubray JM, Martin D, de Lonlay P, Touati G, Poggi-Travert F, Bonnet D, Jouvet P, Boutron M, Slama A, Vianey-Saban C, Bonnefont JP, Rabier D, Kamoun P, Brivet M.
Department of Pediatrics, Hopital Necker Enfants-Malades, Paris, France.

In a personal series of 107 patients, we describe clinical presentations, methods of recognition and therapeutic management of inherited fatty acid oxidation (FAO) defects. As a whole, FAO disorders appear very severe: among the 107 patients, only 57 are still living. Including 47 siblings who died early in infancy, in total 97 patients died, of whom 30% died within the first week of life and 69% before 1 year. Twenty-eight patients presented in the neonatal period with sudden death, heart beat disorders, or neurological distress with various metabolic disturbances. Hepatic presentations were observed in 73% of patients (steatosis, hypoketotic hypoglycaemia, hepatomegaly, Reye syndrome). True hepatic failure was rare (10%); cholestasis was observed in one patient with LCHAD deficiency. Cardiac presentations were observed in 51% of patients: 67% patients presented with cardiomyopathy, mostly hypertrophic, and 47% of patients had heart beat disorders with various conduction abnormalities and arrhythmias responsible for collapse, near-miss and sudden unexpected death. All enzymatic blocks affecting FAO except CPT I and MCAD were found associated with cardiac signs. Muscular signs were observed in 51% of patients (of whom 64% had myalgias or paroxysmal myoglobinuria, and 29% had progressive proximal myopathy). Chronic neurologic presentation was rare, except in LCHAD deficiency (retinitis pigmentosa and peripheral neuropathy). Renal presentation (tubulopathy) and transient renal failure were observed in 27% of patients. The diagnosis of FAO disorders is generally based on the plasma acylcarnitine profile determined by FAB-MS/MS from simple blood spots collected on a Guthrie card. Urinary organic acid profile and total and free plasma carnitine can also be very helpful, mostly in acute attacks. If there is no significant disturbance between attacks, the diagnosis is based upon a long-chain fatty acid loading test, fasting test, and in vitro studies of fatty acid oxidation on fresh lymphocytes or cultured fibroblasts. Treatment includes avoiding fasting or catabolism, suppressing lipolysis, and carnitine supplementation. The long-term dietary therapy aims to prevent periods of fasting and restrict long-chain fatty acid intake with supplementation of medium-chain triglycerides. Despite these therapeutic measures, the long-term prognosis remains uncertain.

Arch Dis Child Fetal Neonatal Ed. 1997 May.
Fat digestion in the neonate.
Manson WG, Weaver LT.
Department of Child Health, Royal Hospital for Sick Children, Glasgow. [ Free full text ]

Excerpts from the full text article:

Introduction

Fats are essential components of the diet, and have a critical role in the growth and development of the neonate. Far from being simply compact sources of energy (providing 40-50% of calorie requirements), they are also integral constituents of neural and retinal tissues.1 2 Dietary fats come in three forms: triacylglycerols; phospholipids; and cholesterol esters, all of which contain fatty acids esterified to alcohols.

The infant consumes fats largely as triacylglycerols, which need to be broken down by enzymes in the upper gastrointestinal tract before absorption. Compared with adults, however, the newborn infant's exocrine pancreas is "immature," secreting only small amounts of lipase even in response to secretagogues.3 4 How the neonate digests fats, and what part they play in neurodevelopment5 is of growing importance, particularly when preterm infants of ever shorter gestation are surviving into adulthood.

Structure, nomenclature and properties of fatty acids

Fatty acids are composed of carbon-carbon (C-C) chains with a carboxylic acid group (-COOH) at one end and a methyl group (-CH3) at the other. The longer the C-C chain, the more concentrated the energy source, but the more difficult the fatty acid is to metabolise. Human milk contains predominantly medium and long chain fatty acids (C:10 to C:22), but other foods contain fatty acids with longer and shorter chains.

Fatty acids are named according to the number of carbon atoms which form the chain and the number of double bonds between them. Thus palmitic acid, which has 16 carbon atoms and no double bonds, is 16:0. Alternatively, the Greek derivation is usedhexadecanoic acid. The carbon atoms are labelled from the carboxylic acid end (fig 1). Either the carboxyl carbon is labelled C1, followed by carbons C2, C3, C4, etc. in sequence, or the first carbon after the carboxyl group is labelled "alpha" (C) and so on, through the Greek alphabet. The last carbon in the chain is referred to as the "omega" carbon (C). The position of the double bonds is denoted either by the number of the first carbon in each bond, counting from the carboxyl end (so -linolenic acid is 9,12,15-octadecatrienoic acid), or by reference to the number of carbon atoms from the "" end where the first double bond is found (so -linolenic acid is 18:3, -3, sometimes written as to 18:3, n-3). The latter is becoming the more widely used notation as it is more physiologically compatible.

Most fatty acids are bound as esters to a glycerol molecule, to form triacylglycerols (fig 1), more commonly but less correctly called triglycerides. In this form they are hydrophobic: they do not dissolve in, or mix well with, water, and therefore they have an important role by binding to, and thus aiding, the transport of fat soluble vitamins. However, this hydrophobia means that they provide a concentrated energy source compared with carbohydrates (9 kcal/g vs 4 kcal/g) which can bind up to 2 g of water for each gram of carbohydrate. Fatty acids are also bound as esters to cholesterol (a precursor of steroid hormones and bile salts), and to phosphate containing alcohols as phospholipids. These are "ambiphilic," with one end hydrophobic and the other hydrophilic, making them ideally suited to form membranes at the interface between aqueous and fat layers (fig 2). Unsaturated fatty acids are usually long chain (>C:16). One or more of the C-C bonds is a double bond and the molecule is therefore not "saturated" with hydrogen. They are more rigid and require an extra metabolic step to break the double bond and to "saturate" the molecule before oxidation. Such fatty acids tend to be used other than for energy: they are essential constituents of the growing brain and retina and precursors of the prostaglandins.1 2 6

To be assimilated, the hydrophobia of dietary fatty acids must be masked so that they can mix with water. In milk they are found in fat globules which contain triacylglycerols surrounded by a membrane formed of ambiphilic phospholipids and cholesterol esters, with their lipophilic ends pointing inwards and their hydrophilic ends outwards (fig 2). These globules can mix with water to form an emulsion. However, if left to stand, being less dense than water, they rise to form a fat layer above an aqueous layer.

Triacylglycerols, cholesterol esters, and phospholipids have an important role in the nutrition of the neonate. In this review we will discuss the assimilation of triacylglycerols and fatty acids in early life, with particular reference to how they are digested in the gastrointestinal tract of the infant.

Human milk

Human milk is a complex mixture of nutrients and non-nutritional factors which provide nourishment and aid the growth and development of the baby. Milk is the sole food for most newborn mammals and it must, therefore, contain a complete and sufficient supply of fluid and nutrients. Milk supplies energy (fat and carbohydrate), protein, vitamins, minerals, immunoproteins, trophic factors and other bioactive substances which play a part in helping the newborn adapt to extrauterine life.7

The fatty acids in human milk have single, unbranched chains with an even number of carbon atoms and varying numbers of double bonds. Small amounts of branched and cyclic fatty acids, and fatty acids with odd numbers of carbon atoms, are also found: these are thought to derive from maternal dietary intake of such fats and do not seem to be of nutritional importance to the infant.8-10 Chain length varies largely between 10 to 22, but fatty acids of 8 and 24 carbon atoms have been found. Fatty acids occur in different ratios which meet the various nutritional requirements of the neonate for them. Table1 shows the relative concentrations of some fatty acids in mature human milk and, for comparison, in unmodified cow's milk.11

Ninety nine per cent of fatty acids in milk are in the form of triacylglycerols.8-10 12-15 A very small proportion (<0.1%) occurs as diacylglycerols and free fatty acids, but this may be an artefact from processing the milk for assay. The other 1% occurs as cholesterol esters (10-15 mg/dl) and phospholipids (15-20 mg/dl).

The fat content of human milk changes during early lactation. It increases from 2.0 g/dl in colostrum to 4.9 g/dl in mature milk, reflecting the increasing energy requirement of the growing infant. However, the fat content of milk also varies during feeds, from 3.0 g/dl in midday foremilk to 4.0 g/dl in midday hindmilk, and during the day, from 3.0 g/dl in early morning milk to 4.5 g/dl in evening milk.8 9

During the transition from colostrum to mature milk (table 2),10 the proportions of cholesterol and phospholipid relative to total fat content fall (1.3% down to 0.4%, and 1.1% to 0.6%, respectively). However, this is almost entirely due to an increase in concentration of triacylglycerols rather than to a decrease in concentration of the other two lipids: phospholipids actually increase in concentration from 22.4 to 29.2 mg/dl.

Humans can elongate fatty acids to extend chain lengths and, in some circumstances, can desaturate the chain to make double bonds. However, double bonds cannot be inserted beyond the C9 carbon and so a supply of -3 and -6 fatty acids (such as linoleic (18:2, -6) and -linolenic (18:3, -3) acids) is required to synthesise arachidonic (20:4, -6; AA) and docosahexaenoic (22:6, -3; DHA) acids. These are essential structural components of neural tissue, and also precursors of the eicosanoids.5 In the neonate these enzyme systems (elongases and desaturases) are not fully developed. Therefore, although in adults linoleic and -linolenic are regarded as the only essential fatty acids, the newborn infant also has a dietary requirement for AA and DHA.

Triacylglycerols are stereo-specific and the three ester bonds are not equally susceptible to hydrolysis by lipase enzymes. Fatty acids are not randomly distributed among the three stereo-specific numbering (Sn) positions, but are found selectively placed to provide the ideal mixture of fatty acids and monoacylglycerols for the neonate (table 3)16: for example, a relative abundance of 16:0 (palmitic acid) at the Sn2 position provides the monoacylglycerol 2-palmitoyl-glycerol which is a potent antimicrobial, and with palmitate in this position, the absorption of other fatty acids may increase.17 18

Lipases

The lipases which act in the infant gut can be categorised into preduodenal, pancreatic, and breast milk lipases.

Preduodenal lipase

There is uncertainty about the nature and origin of preduodenal lipases. The fundus of the stomach and von Ebner's glands around the circumvillate papillae of the tongue have both been proposed as sources.19-21 The evidence for a "gastric lipase" is that, when incubated with triacylglycerol, samples of gastric fundus release free fatty acids. The samples used have included gastric biopsy specimens from all ages, and pieces of stomach obtained from babies dying of cot death, stillborn babies, and aborted fetuses. Gastric lipase can be found in samples from fetuses as early as 18 weeks of gestation, attain significant levels of activity by 27 weeks,19 but do not reach normal adult levels until the first few months of age. However, this gastric lipase activity may well derive from lipase secreted by the tongue which has been adsorbed on to the gastric mucosa and has not been washed off specimens during preparation.22

Lipase activity has also been detected in the upper oesophageal pouches of babies with congenital oesophageal atresia.23 It is found in the tongue of the rat fetus at 20 days,24 and there is evidence for its presence in the glands of von Ebner in humans.20 However, it has been argued that lipase found in oesophageal pouches represents reflux of gastric secretions through the tracheo-oesophageal fistula, present in many of the babies studied. Moreau et al21 found no lypolytic activity in biopsy specimens of tongue, pharynx, and oesophagus, taken at endoscopy and from transplant donors, suggesting that preduodenal lipase originates from the stomach alone. Both lingual and gastric lipases may exist, but the extent to which each contributes to preduodenal lipolysis remains unclear. These two moieties of lipase, lingual and gastric, seem to have similar molecular weights, structures, and conditions for action13 15 25 and hereafter they will be referred to collectively as "preduodenal" lipase.

Preduodenal lipase consists of a polypeptide chain of 379 amino acid residues with a molecular weight of around 43000 Daltons.25 It embeds itself in the phospholipid surface layer of the milk fat globule and digests the lipids within(fig 2). It acts preferentially at the Sn3 position, hydrolysing only very small amounts of fatty acids at the Sn1 and Sn2 positions. When Hamosh et al26 measured the free fatty acids released in the neonatal stomach by preduodenal lipolysis, they found a predominance of medium chain saturated and long chain unsaturated fatty acids, and concluded that preduodenal lipase preferentially hydrolysed these fatty acids. However, another study27 has questioned this conclusion, suggesting that an abundance of such fatty acids at the Sn3 position and preference of preduodenal lipase to hydrolyse fatty acids at this position would explain to the findings described by Hamosh et al.26

Preduodenal lipase has a low optimal pH (2.5-6.5), and is resistant to the acid conditions of the stomach and to gastric proteases. It does not require cofactors or bile salts and is rapidly inactivated by pancreatic trypsin and therefore ceases to be active when the milk bolus passes into the duodenum.25 However, in cystic fibrosis, where pancreatic function and hence trypsin concentrations are low, its action may continue in the duodenum and compensate to some extent for depressed pancreatic lipase activities.28

Preduodenal lipase has an important role in the initiation of lipolysis in the stomach, with the liberation of short and medium chain and -3 and -6 fatty acids, and the preparation of the milk emulsion for further lipolysis by pancreatic and breast milk lipase.13 26 29

Pancreatic lipase

Lipase is secreted by the pancreas from approximately 30 weeks of gestation onwards.30 However, in both term and preterm infants it is present at very low concentrations until well into the first year of life.3 It is a polypeptide of 449 amino acid residues, has a molecular weight of approximately 50000 Daltons,25 an optimal pH of 6.5-8.0 and an absolute requirement for colipase and bile salts. It has little action on soluble esters, preferring a lipid/water interface,31 and hydrolyses triacylglycerols at the Sn1 and Sn3 positions, liberating 2-monoacylglycerols and free fatty acids.

Pancreatic lipase by itself is not very effective at hydrolysing triacylglycerols found in milk in vitro. However, if milk is predigested with preduodenal lipase, there is a 20-fold increase in the release of free fatty acids compared with that from milk digested with pancreatic lipase alone.13 It has been suggested that as much as 25% of free fatty acids are hydrolysed by preduodenal lipase.29

Breast milk lipase

It has been known since the turn of the century that human milk has the capacity to hydrolyse esters.32 A breast milk lipase was first described in 195333 and since then, human milk lipoprotein lipase has been detected and characterised.

Because of its absolute requirement for bile salts, breast milk lipase is more commonly referred to as bile salt stimulated lipase (BSSL). BSSL is present in term and preterm milk and is found in the highest concentrations in the colostrum of mothers of preterm infants. Although the amount of lipase secreted by different women varies, each mother produces relatively constant concentrations of BSSL until weaning.34

BSSL has 722 amino acid residues and a molecular weight of around 90 000 Daltons. Differences in reported molecular weights may be explained by differences in glycosylation of the enzyme. BSSL shares little homology with other human lipases, but sequences are similar to those in esterases such as acetyl choline esterase. This may explain, in part, the non-specific action of BSSL compared with other lipases.25

BSSL is present in the aqueous fraction of the milk emulsion and does not hydrolyse triacylglycerol, which is held inside the milk fat globule, until the milk reaches the duodenum. BSSL is activated by primary bile salts (cholate and chenodeoxycholate) in two ways: firstly, the size of the globules is reduced by the action of bile, increasing the surface area of the globules on which BSSL can act; secondly, the bile salts bind with BSSL in such a way as to facilitate hydrolysis of triacylglycerols. BSSL is non-specific in its action on the triacylglycerol molecule: it hydrolyses fatty acids at all three positions (Sn1, 2, and 3) to release glycerol and free fatty acids.

With growing evidence that long chain polyunsaturated fatty acids have an important role in neonatal development, it is possible that, in the presence of low concentrations of pancreatic lipase, the action of BSSL may be fundamental to the optimal nutrition and neurofunction of neonates. It is important to note that pasteurising or boiling donor expressed breast milk reduces fat absorption to 73% and 64%, respectively, compared with raw human milk.35

Gastrointestinal digestion and absorption of milk lipids

The digestion and absorption of fat in the gastrointestinal tract occurs in several stages. After ingestion milk is further emulsified in the stomach: gastric motility and acidity act on the milk to decrease the size of the fat globules. This promotes the action of preduodenal lipase, resulting in partial digestion of lipids. The milk then enters the duodenum as a coarse chyme and is mixed with bile, which further reduces the size of the milk globules and promotes hydrolysis. The smaller fat globules present a larger surface area relative to volume for the action of pancreatic lipase and BSSL. At a critical concentration bile salts aggregate to form micelles, which have a highly polar surface and a non-polar, hydrophobic core. They solubilise the products of hydrolysis (glycerol, monoacylglycerols, and the hydrophobic free fatty acids) to form mixed micelles. The hydrophobic core attracts other non-polar molecules such as fat soluble vitamins. The resultant small globule, with its polar, hydrophilic surface, then undergoes absorption. The mixed micelle comes into contact with the brush border of the small intestine and free fatty acids, and acylglycerols diffuse into the mucosal cell. In the endoplasmic reticulum fatty acids bind with fatty acid binding protein and triacylglycerols are resynthesised. These are released into the circulation as chylomicrons and pass via the portal system to the liver where they are metabolised. The short and medium chain fatty acids are used for energy, either being oxidised immediately to carbon dioxide and water, or being transferred to fat stores. Most longer chain unsaturated fatty acids are used, either as they are or after further desaturation and/or elongation, for the synthesis of cell membranes and bioactive molecules, such as prostaglandins.

Conclusions

Most of our understanding of the digestion of milk fat by the newborn infant is based on extrapolation from adult studies, experiments performed on neonatal mammals, or measurements of the lipolytic activity of secretions from human fetuses and infants in vitro. Fat balance studies have provided some measure of the efficiency of lipid digestion and absorption in the newborn, but there are few published studies of the functional capacity of the neonate to digest fat.

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Fats labelled with 13C have been used to assess pancreatic function in adult health and disease,36 and in children to assess fat digestion in cystic fibrosis.37 The substrate used in these studies was a "mixed triacylglycerol" (MTG) with 13C labelled octanoic acid in the Sn-2 position (fig 3). The stearic acids on the Sn-1 and Sn-3 positions are hydolysed by lipases, releasing labelled monoglyceride which is absorbed and oxidised, releasing 13CO2. The percentage dose of 13C recovered after 6 hours (PDR) is calculated and used as an expression of functional fat digestion.38 The choice of octanoic acid (which is rapidly absorbed and oxidised39-43) as the fatty acid in the Sn2 position ensures that the rate limiting step is the digestion of the two long chain fatty acids in the Sn1 and Sn3 positions. As human milk contains very little, if any, octanoic acid,9 the labelled tracer is not significantly diluted by unlabelled substrate.

Fat digestion in infancy has also been studied using this technique. Hoshi et al44 studied five term neonates at 3 days of age and five "growing preterm infants," using 13C-trioctanoyl-glycerol (trioctanoin) as a substrate. They reported mean PDRs of 53% in term neonates and 46% in the preterm group, values significantly higher than those obtained in older children by McClean et al,45 who reported a mean PDR of 24%. More recently, MTG (1,3-distearyl, 2-13C-octanoyl glycerol) has gained popularity over trioctanoin because the combination of fatty acids on the triacylglycerol is specific for pancreatic lipase. Using the MTG breath test to measure lipase activity in preterm infants of 27 to 35 weeks gestational age and aged 14 to 55 days, van Aalst46 reported a mean PDR of 25%. When we performed MTG breath tests on neonates aged 1 to 3 days, we recorded a mean PDR of 16% (range of 0-32%). By 7-21 days this had increased to 23% and by late infancy to 29%.47

Together, these studies suggest that the capacity of the term newborn to digest fat during early neonatal life varies widely, ranging from nil to near normal adult levels. Thereafter, it increases in both term and preterm infants. In some children with cystic fibrosis and depressed pancreatic function PDR can be zero,37 suggesting that the MTG is an appropriate substrate with which to measure pancreatic lipase acitivity.

"Designer lipids," triacylglycerols with labelled fatty acids in different combinations and at different sites, offer a means of studying the changing capacity of infants to digest fats in early life. They can be tailor-made to measure the activity of specific enzymes in the gastrointestinal tract so that the relative contributions of preduodenal, pancreatic, and breast milk lipases to overall fat digestion can be determined.

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The significant difference, not only in the relative proportions of fatty acids in human and cow's milk, but also in their distribution on the triacylglycerol molecule, has major implications for the neurodevelopment of the newborn.5 Milk is the only source of essential fatty acids for the growing infant, and it is now recognised that feeding babies on formulas based on cow's milk may be associated with deficient neurodevelopment and retinal function in infancy48 and, in preterm infants, reduced intelligence quotient in later childhood.49 It is therefore vital that we understand more fully the fate of ingested lipids, what regulates their digestion, and as a result, which fatty acids are available for absorption and deposition in neural tissues.

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