Intracellular Movement And Biosynthesis Of Lipids

Fatty Acids

SAFA are biosynthesized in the extramitochondrial compartment by a group of enzymes known as FA synthetases. Compared with many animal species, human FA synthesis occurs predominantly in the liver and is much less active in adipose tissue. The FA biosynthetic pathway is almost identical in all organisms examined to date. The starting point is acetyl-CoA. Acetyl-CoA and oxaloacetate are cleaved from citrate, which is transported from the mitochondria. The first reaction in the FA biosynthetic pathway proper is the conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase, which is rate limiting for FA synthesis. Acetyl-CoA then combines sequentially with a series of malonyl-CoA molecules as follows:

Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14H+ ® C16:0 (palmitic acid) + 7 CO2 + 8 CoASH + 14 NADP+ + 6 H20

In mammals, complete de novo synthesis results in C16:0. Other FA can be formed from C16:0 by chain elongation via microsomal malonyl-CoA-dependent elongase. Mammals possess a series of desaturases and elongases to generate long-chain PUFA from the metabolism of C16:0, C18:0, C18:2n-6, and C18:3n-3 (Fig.:.4:6). These reactions occur predominantly in the endoplasmic reticulum membranes. Desaturase reactions are catalyzed by membrane-bound desaturases with broad chain-length specificity, including D9, D6, D5, and D4 fatty acyl-CoA desaturases. These are involved in the desaturation of the C16:1n-7, C18:1n-9, C18:2n-6, and C18:3n-3 families. The D4 desaturation required for formation of C22:6n-3 from C22:5n-3, and C22:5n-6 from C22:4n-6, respectively, involves three steps. These steps require an elongation reaction followed by membrane (microsomal) desaturation and shortening in peroxisomes. The desaturase enzymes are highly specific for the position of the double bond. The FA desaturase system involves three integral components: the desaturase, NADH-cytochrome b 5 reductase, and cytochrome b5, which are constituents of microsomal membranes. Desaturases require electrons supplied mostly by NADH-cytochrome b5 reductase in addition to the activated substrate in the form of acyl-CoA.

Precursors for the n-7 and n-9 families of PUFA are MUFA that are synthesized via microsomal D9 oxidative desaturation of C16:0 and C18:0 to form C16:1n-7 and

C18:1n-9, respectively (Fig. 4:..§)- Additional double bonds can be introduced into existing MUFA C16:1n-7 and C18:1n-9 and also into C18:2n-6 via D 6 desaturase (Fig.4.6). Until recently, humans and other mammals were thought incapable of synthesizing long-chain n-3 (C18:3n-3) and n-6 (C18:2n-6) EFA. Recent studies, however, suggest that C18:2n-6 and C18:3n-3 can be synthesized in humans and other mammals via elongation of the dietary precursors C16:2n-6 and C16:3n-3, respectively (78). Edible green plants can contain up to 14% C16:2n-6 and C16:3n-3 (78). In a practical sense, a dietary supply of EFA is still important, since humans likely do not obtain enough 16-carbon precursors.

Figure 4.6. Effects of desaturase and elongase on essential fatty acids.

Figure 4.6. Effects of desaturase and elongase on essential fatty acids.

In mammals, FA from the n-3 and n-6 FA cannot be interconverted because of a lack of D12 or D15 desaturase enzymes, although such interconversions can take place in plants. D6 Desaturase is the regulatory enzyme in these reactions and requires an n-9 cis double bond. Hence, trans FA, such as C18:1n-9 trans, formed either by rumen bacteria or by chemical hydrogenation of FA with cis double bonds, cannot be desaturated by this enzyme. The n-3, n-6, and n-9 FA families compete with each other, especially at the rate-limiting D6 desaturase step. In general, desaturase enzymes display highest affinity for the most highly unsaturated substrate. The order of preference is a-linolenic family (n-3) > linoleic family (n-6) > oleic acid family (n-9) > palmitoleic acid family (n-7) > elaidic acid family (n-9, trans). Competition also exists among the families of PUFA for the elongase enzymes and for the acyl transferases involved in formation of PL.

Because of the competitive nature of FA desaturation and elongation, each class of EFA can interfere with the metabolism of the other. This competition has nutritional implications. An excess of n-6 EFA will reduce the metabolism of C18:3n-3, possibly leading to a deficit of its metabolites, including eicosapentanoic acid (C20:5n-3). This is a matter of concern in relation to infant formulas, which may contain an excess of C18:2n-6 with no balancing of n-3 EFA. Conversely, as long-chain n-3 EFA markedly decrease D6 desaturation of C18:2n-6, excessive intake of fish oils could lead to impairment of C18:2n-6 metabolism and a deficit of n-6 EFA derivatives. High doses of fish oil in humans can cause a large reduction in the levels of C20:3n-6 in plasma PL, with a smaller effect on C20:4n-6 content ( 79). Although C18:1n-9 can inhibit D6 desaturase activity, high dietary intakes are necessary. In the presence of C18:2n-6 or C18:3n-3, little desaturation of C18:1n-9 occurs. During EFAD, C20:3n-9 is synthesized from C18:1n-9 because of the nearly complete absence of competitive effects of n-3 and n-6 EFA. The presence of C20:3n-9 in tissues instead of C20:4n-6, C20:5n-3, and C22:6n-3 indicates EFAD, which reverses on EFA feeding ( 80). In the catalytic hydrogenation of vegetable oils and fish oils for the production of some margarines and shortenings, a variety of geometric and positional isomers of unsaturated FA are formed in varying amounts. After absorption, these isomers may compete with the EFA and endogenously synthesized FA for desaturation and chain elon-gation.

In a phenomenon called retroversion, very long-chain C22 PUFA present in marine oils may be shortened by two carbons with concomitant saturation of a double bond. For example, C22:6n-3 is converted to C22:5n-3 and to C20:5n-3 (81). This peroxisomal pathway is also active in converting C22:5n-6 into C20:4n-6 ( 82). As a result of competition among various PUFA families for desaturases, elongases, and acyl transferases, and because of retroversion, a characteristic pattern of end products accumulates in tissue lipids for each family. Hence, the major PUFA product for the palmitoleate n-7 family is C20:3n-7; for the oleate n-9, C20:3n-9; and for linoleate, C20:4n-6 and some C20:3n-6. The most common products for the n-3 fatty acid family are C20:5n-3 and C22:6n-3.

The efficiency of the multistage synthesis of PUFA is unclear in the human. It has been suggested that activities of the various required desaturase and elongase enzymes differ with developmental stage or pathologic state. Regulation of desaturase activity could be of biologic importance, since the higher homologues of EFA are physiologically important regulatory metabolites.

Dietary factors and hormonal status can influence desaturase activities. Fat-free diets result in increased D 5 and D6 desaturation, which may reflect a homeostatic response to maintain membrane fluidity (83). Protein and EFAD increase D6 desaturase activity; conversely, low-protein diets and alcohol consumption decrease D 6 activity. Although glucose refeeding after a fast induces D6 desaturase activity, a glucose-rich diet actually decreases enzymatic activity. Insulin stimulates D 6 desaturase activity; activity is depressed by glucagon, epinephrine, glucocorticoids, and thyroxines. Diabetes also depresses D 6, D5, and D4 desaturase activities, which are restored by insulin injection (84). Zinc may also play a role in the regulation of D6 desaturase activity, as the dermal and growth effects of EFA and zinc deficiency are similar (85). This concept is supported by observations that administration of C18:3n-6, which bypasses the D 6 desaturase step, corrects most of the symptoms of zinc deficiency, whereas administration of C18:2n-6 has no effect. As the typical Western diet contains sufficient C20:4n-6, obtained from meat and dairy products, those with decreased desaturase activity could suffer from a deficiency of C20:3n-6, the precursor of the PG "1" series. Some authors have suggested that certain individuals may have increased need for EFA derivatives because of a disease condition, aging, or a metabolic block in desaturase activity. Evening primrose, borage, and black current seed oils contain C18:3n-6 that bypasses the step requiring D 6 desaturase and have been used therapeutically for a variety of clinical conditions, including psoriasis (86).

Cholesterol

Current evidence indicates that three distinct pathways modulate the intracellular transmission of CH. Separate translocational systems exist for endogenously synthesized and LDL-derived exogenous CH. A third transport system also exists for CH destined for steroid synthesis.

CH biosynthesis represents a major vector in the total body CH supply in humans, with up to about 75% being synthesized during consumption of the typical North American diet. Animal studies demonstrate that even though all organs incorporate acetate into sterol, the liver is the primary biosynthetic organ ( 8.7). Conversely, in humans, it has been estimated that the net contribution of liver biosynthesis does not exceed 10% of total CH biosynthesis. The role of extrahepatic organs in human cholesterogenesis remains undefined.

Acetate can be converted into mevalonic acid by a sequence of reactions starting with acetate + CoA + ATP ® 1A acetyl-CoA + PP + AMP. However, most of the acetyl-CoA used for sterol synthesis is not derived from this reaction but rather is generated within the mitochondria by b-oxidation of FA or oxidative decarboxylation of pyruvate. Pyruvate is converted into citrate, which diffuses into the cytosol and is hydrolyzed to acetyl-CoA and oxaloacetate by citrate-ATP lyase:

Citrate + ATP + CoA ® 1A acetyl-CoA + oxaloacetate + ADP + H2O

Citrate participating in this reaction acts as a carrier to transport acetyl carbon across the mitochondrial membranes, which are impermeable to acetyl-CoA. Subsequently, in the cytosol, acetyl-CoA is converted into mevalonate:

2 aictyl CoA acctoacctvl CoA + CoA Aceioacetyl-CoA + acetyl CoA + —> HMG'CoA + CoA

Mevalonic acid is phosphorylated, isomerized, and converted to geranyl- and farnesyl-pyrophosphate, which in turn form squalene. Squalene is then oxidized and cyclized to a steroid ring, lanosterol. In the last steps, lanosterol is converted into CH by the loss of three methyl groups, saturation of the side chain, and a shift of the double bond from D8 to D5. During the later stages of CH biosynthesis, intermediates are bound to a sterol carrier protein.

CH biosynthesis in humans is sensitive to a number of dietary factors. Adding CH to the diet at physiologic levels results in modest increases in circulating CH levels, with a mild reciprocal inhibition of synthesis (28, 50). Dietary fat selection exhibits a more pronounced influence on human cholesterogenesis, as consumption of polyunsaturated fats is associated with higher biosynthesis than other plant or animal fats. Differences in FA composition and levels of plant sterol levels may both be contributing factors (35). Higher meal frequency reduces biosynthesis rates in humans, which may explain the lower circulating CH synthesis rates seen in individuals consuming more numerous smaller meals (88). Insulin, which is associated with hepatic CH synthesis in animals, may be released in greater amounts when less frequent but larger meals are consumed. Circadian periodicity, with a maximum at night, is tied to the timing of meal consumption. Of dietary factors capable of modifying CH synthesis, energy restriction exhibits the greatest effect. Humans fasted for 24 hours exhibit complete cessation of CH biosynthesis ( 18). How synthesis responds to more minor energy imbalance has not been examined.

There is an emerging view that CH synthesis acts both passively and actively in relation to circulatory CH levels, depending on dietary perturbation. Passively, the liver responds to high CH levels through LDL receptor-mediated suppression of synthesis ( 42). The modest suppression in the face of increasing dietary and circulating levels reflects the limited hepatic contribution to total body production of CH ( 28). Substitution of PUFA for other fats results in a decreased ratio of hepatic intracellular free CH to esterified CH, which in turn upregulates both LDL receptor number and cholesterogenesis. In both of these ways, CH synthesis responds passively to external stimuli. In contrast, nonhepatic synthesis is less sensitive to dietary CH level and fat type, while together with hepatic synthesis, nonhepatic synthesis is more responsive to synthesis pathway substrate availability (89). In this manner, several dietary factors actively modify CH synthesis and levels. Such differential sensitivity may explain the more pronounced decrement in Ch synthesis and levels occurring after energy deficit in humans.

CH serves as a required precursor for other important steroid compounds, including sex hormones, adrenocorticoid hormones, and vitamin D. Steroidal sex hormones, including estrogen, androgen, and progesterone, involve removal of the CH side chain at C-17 and rearrangement of the double bonds in the steroid nucleus. Corticosteroid hormone production involves similar rearrangements of the CH molecule. 7-Dehydrocholesterol is the precursor of cholecalciferol (vitamin D) formed at the skin surface through the action of ultraviolet irradiation. Steroid hormone metabolites are excreted principally through the urine. It is estimated that humans convert about 50 mg/day of CH to steroid hormones.

Vertebrates cannot convert plant sterols to CH. However, insects and prawns can transform phytosterols into steroid hormones or bile acids through a CH intermediate.

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