LCAT activity

HDL is synthesized and secreted from both liver and intestine (Figure 25—5). However, apo C and apo E are synthesized in the liver and transferred from fiver HDL to intestinal HDL when the latter enters the plasma. A major function of HDL is to act as a repository for the apo C and apo E required in the metabohsm of chylomicrons and VLDL. Nascent HDL consists of discoid phosphohpid bilayers containing apo A and free cholesterol. These hpoproteins are similar to the particles found in the plasma of patients with a deficiency of the plasma enzyme lecithimcholesterol acyltransferase (LCAT) and in the plasma of patients with obstructive jaundice. LCAT—and the LCAT activator apo A-I— bind to the disk, and the surface phosphohpid and free cholesterol are converted into cholesteryl esters and... 

LCAT activity is associated with HDL containing apo A-L As cholesterol in HDL becomes esterified, it cre-... 

This protein is found in plasma of humans and many other species, associated with HDL. It facilitates transfer of cholesteryl ester from HDL to VLDL, IDL, and LDL in exchange for triacylglycerol, relieving product inhibition of LCAT activity in HDL. Thus, in humans, much of the cholesteryl ester formed by LCAT finds its way to the hver via VLDL remnants (IDL) or LDL (Figure 26-6). The triacylglycerol-enriched HDL2 delivers its cholesterol to the hver in the HDL cycle (Figure 25-5). 

LCAT acts preferentially on lipids transported by HDL (so-called a-LCAT activity), but also on lipids transported by apoB-containing lipoproteins (so-called jS-LCAT activity) [58, 85]. In practice, LCAT activity is measured either as the activity required to esterify radioactive cholesterol that has been exogenously incorporated into native HDL or into artificial HDL-like particles (a-LCAT activity) or which has been equilibrated with endogenous lipoproteins of the plasma sample (cholesterol esterification rate, CER) [21, 58, 85]. Several variations of these assays have been reported, some of which are available as commercial test kits (e.g., Roar Biomedical, New York, USA). In addition, LCAT concentration can be determined by either laboratory-made tests or by a commercial ELISA kits [57]. However, the decrease in LCAT concentration is difficult to judge since it also decreases secondary to HDL deficiency due to causes other than LCAT deficiency. Plasma from patients with LCAT deficiency fails to esterify radioactive cholesterol provided by any substrate. By contrast, plasmas of patients with fish-eye disease show a near-normal cholesterol ester-fication rate but have a selective inability to esterify radioactive cholesterol provided to plasma with native HDL or reconstituted HDL (a-LCAT activity) [58, 85]. 

For -LCAT activity the apoA-I proteoliposome emulsion is prepared by evaporating 260 pi of 5 mg/ml egg yolk phosphatidylcholine, 150 pi of 1 mg/ml unesteri-fied cholesterol, and 3 pi of 21 Ci/mmol [7-3H(N)]-cholesterol. The dried lipids are dissolved in 125 pi pure ethanol and injected into 10 ml of analysis buffer and vor-texed. The emulsion is concentrated by ultrafiltration to less than 2.5 ml and then filled up to 2.5 ml. A 300-pL aliquot of this emulsion is incubated with 75-150 mg of apoA-I and 1.1 ml analysis buffer. The optimal amount of apoA-I varies from lot to lot and has to be optimized using normal plasma samples. 

Patients with classical LCAT deficiency fail to esterify cholesterol in any substrate and hence have both an undetectable or very low cholesterol esterification rate and a-LCAT activity. Patients with fish-eye disease usually have a normal cholesterol esterification rate and a selective a-LCAT deficiency. 

Considerable amounts of LCAT are carried by HDL therefore a-LCAT activity is also secondarily reduced in other forms of familial HDL deficiency. Notably, this partial LCAT deficiency has been repeatedly documented in forms of apoA-I deficiency due to structural defects in apoA-I. However, despite secondary LCAT deficiency these patients have a normal unesterified cholesterolitotal cholesterol ratio [35]. 

Funke H, Eckardstein A von, Pritchard PH, Albers JJ, Kastelein JJ, Droste C, Assmann G (1991) A molecular defect causing fish eye disease an amino acid exchange in lecithin-cholesterol acyltransferase (LCAT) leads to the selective loss of alpha-LCAT activity. Proc Natl Acad Sci U S A 88 4855-4859... 

ApoD is found in association with LCAT and with apoA-I in the HDL fraction. Albers et al. used a specific antibody to apoD to remove all apoD by immunoadsorption chromatography from plasma about 64% of LCAT activity and 11% of apoA-I were also removed from plasma (A14). Purified apoD has an apparent Mr of 32,500, and appears as three isoforms on isoelectric focusing (pi 5.20, 5.08, and 5.00) (A14). An HDL apolipoprotein, Mr 35,000, has been thought to be apoD, and to be a cholesteryl ester transfer protein (i.e., to transfer newly synthesized esterified cholesterol from HDL to LDL) (C8). Cholesteryl ester transfer activity in plasma was removed by polyclonal immunoglobulin to apoD (C8, F10). However, Morton and Zilversmit (M41) were able to separate apoD and lipid transfer protein (i.e., the cholesteryl ester transfer protein, or lipid transfer protein I) by chromatography, and they showed that the removal of apoD from plasma by precipitation with specific antisera did not remove any lipid transfer activity. Albers et al. (A14) also showed that immunoadsorption with antibody specific for apoD removed all the apoD from plasma without removing any cholesteryl ester transfer activity. 

The major function of LTP-I in human plasma may be to distribute es-terified cholesterol from the HDL fraction, where cholesterol is esterified, to other lipoprotein fractions. LCAT activity is responsible for the production of some 50-100 nmol esterified cholesterol per milliliter of plasma per hour (Gil). The concentration of esterified cholesterol in human HDL is about 1000 nmol/ml of plasma. Only 0.5-1.0% of HDL apoprotein is removed from plasma per hour (B41), probably mainly in intact HDL particles. If so, then the uptake of HDL particles can account for the removal from plasma of only about 10-20% of the esterified cholesterol formed in HDL in the LCAT reaction. 

LCAT in the rat appears to be produced by the liver (B48, Nil, Oil). Although there seems to be no direct evidence of production by the liver in man, the hepatic origin of LCAT is suggested by the marked reduction in LCAT activity associated with liver diseases (C23, S51) including viral hepatitis (B37, Til) and cirrhosis (B37, S2, S36). LCAT activity is also said to be reduced in uremia (Sll) and pancreatic carcinoma (S36). The concentration of LCAT in normal human plasma (measured by radioimmunoassay) is about 6 mg/liter (A8). 

LCAT, whereas LCAT activity with HDL2 as a substrate is minimal. Others have confirmed that HDL3 is a better substrate for LCAT than HDL2 (Bll, M28). Hamilton et al. (H5) noted that nascent disk-shaped HDL secreted by rat liver were better substrates for LCAT than mature spherical HDL isolated from plasma. Synthetic discoidal complexes of apoA-I, phosphatidylcholine, and cholesterol were better substrates for LCAT than unilamellar vesicles of phosphatidylcholine and cholesterol, incubated in the presence of apoA-I (M32). 

The rate of cholesterol esterification in plasma is not correlated with HDL concentration (A12, R17, S45, S58, Wl, W2) but is correlated with the concentration of VLDL or triglyceride (A12, P8, R17, S58, T7, Wl, W2). Although HDL is the major substrate for LCAT, VLDL and indirectly LDL are the major recipients of the esterfied cholesterol, transferred (it is thought) by lipid transfer protein. Accumulation of esterified cholesterol in the recipient lipoproteins is associated with a decrease in LCAT activity (C7, Fll, F13) that can be relieved by the addition of recipient lipoproteins but not by addition of LCAT substrate (Fll). Hopkins and Barter (H32, H33) have explained these observations by showing that the depletion of HDL esterified cholesterol by transfer to VLDL enhances the capacity of HDL to act as a substrate for LCAT. 

A12. Albers, J. J., Chen, C.-H., and Adolphson, J. L., Lecithimcholesterol acyltransferase (LCAT) mass its relationship to LCAT activity and cholesterol esterification rate. /. Lipid Res. 22, 1206-1213 (1981). 

Forsythe, W.A., Miller, E.R., Hill, G.M., Romsos, D.R., and Simpson, R.C. 1980. Effects of dietary protein and fat sources on plasma cholesterol parameters, LCAT activity and amino acid levels and on tissue lipid content of growing pigs. J. Nutr. 110(12), 2467-2479. 

At the end of a fifteen-week aerobic exercise program, 19 male participants had a 14% increase in physical performance capacity and a twofold increase in LCAT activity (68). LCAT activity was elevated after seven weeks of exercise, and it continued to Increase significantly throughout the remainder of the exercise program. At present, however, the magnitude of the effect of exercise on LCAT activity is not clear. 

A proposed mechanism by which an exercise-induced increase in LCAT leads to an increase in HDL cholesterol is illustrated in figure 3. An increase in both Apo AI and LCAT activity in response to exercise could lead to Increased esterification of cholesterol in HDL and thereby allows for an increase in the transport of free cholesterol from tissues and other lipoproteins to nascent HDL, and enhanced formation of HDL2. While still speculative, this proposed mechanism deserves attention. 

Figure 3. Proposed yet speculative mechanism by which high-denslty lipoprotein (HDL) increases in response to exercise via Increased lecithin cholesterol acyl transferase (LCAT) activity and/or Increased apolipoproteln A concentration.

Lecithin-cholesterol acyltransferase is a water-soluble plasma enzyme that plays an important role in the metabolism of HDLs by catalyzing the formation of cholesteryl esters on HDLs through the transfer of fatty acids from the sn-2 position of phosphatidylcholine to cholesterol (Jonas, 1986). ApoA-1 is the major cofactor of LCAT in HDLs and reconstituted lipoproteins (Fielding et ai, 1972). Many laboratories have used techniques such as synthetic peptide analogs (Anantharamaiah et ai, 1990a Anantharamaiah, 1986), monoclonal antibodies (Banka et al., 1990), and recombinant HDL particles (Jonas and Kranovich, 1978) to attempt to identify the major LCAT-activating region of apoA-I. 

Although all of the exchangeable apolipoproteins contain amphipathic helices, apoA-I is the superior LCAT activator (Anantharamaiah et ai, 1990a). Further (with the exception to be discussed below), all amphipathic helical peptides studied have an intrinsic upper limit to their ability to activate LCAT, approximately 30% of apoA-I (Anantharamaiah et ai, 1990a Anantharamaiah, 1986). Therefore, simple water-phospholipid interface disruption by amphipathic helices may be necessary for LCAT activation, but is clearly not sufficient. Additional structural features must be involved. 

As shown in Fig. 7, structural analysis suggests the presence, starting at residue 44, of 10 tandem and structurally separate class A or class Y amphipathic helical domains in apoA-1. Several lines of evidence point to the amino-terminal region of these tandem amphipathic helices as the predominant LCAT-activating domain in apoA-I ... 

ApoA-l-specihc monoclonal antibodies have been used in conjunction with synthetic peptides to suggest that part of the LCAT activation domain resides between residues 96 and 111 (Banka et al., 1990). 

Earlier studies failed to identify a major LCAT-activating domain among the four fragments produced by CNBr hydrolysis of apoA-1 (Fielding et al., 1972). It is likely that the functional importance of the 66-120 domain was missed in these earlier studies because two of the three methionines present in apoA-I are found at the end of helix 2 (residue 86) and in the middle of helix 3 (residue 112). It is likely that cleavage at these positions destroyed the conformation of the region required for LCAT activation. 

Banka, C. L., Smith, R. S., Bonnet, D. J., and Curtiss, L. K. (1990). Localization of an Apolipoprotein A-1 Epitope Critical for LCAT Activation, 63rd Sci. Sess. American Heart Association. Dallas, TX. 

---