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Journal of Lipid Research, Vol. 48, 185-192, January 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology
Patient-Oriented Research |
* Departments of Pathology and Physiology, Louisiana State University Health Sciences Center, New Orleans, LA
Department of Medicine, Oregon Health and Science University, Portland, OR
Published, JLR Papers in Press, October 1, 2006.
1 To whom correspondence should be addressed. e-mail: connorw{at}ohsu.edu
ABSTRACT
To study the metabolism of cholestanol in patients with cerebrotendinous xanthomatosis (CTX), we measured the cholestanol absorption, the cholesterol and cholestanol turnover, and the tissue content of sterols in two patients. Cholestanol absorption was 
5.0%. The rapid exchangeable pool of cholestanol was 233 mg, and the total exchangeable pool was 752 mg. The production rate of cholestanol in pool A was 39 mg/day. [4-14C]cholestanol was detected in the xanthomas, but neither [4-14C]cholestanol nor [4-14C]cholesterol was detected in peripheral nerves biopsied at 49 and 97 days after [4-14C]cholesterol given intravenously. Of the 18 tissues analyzed at biopsy and autopsy, the cholestanol content varied from 0.09 mg/g in psoas muscle to 76 mg/g in a cerebellar xanthoma. With the assumption that the cholestanol-to-cholesterol ratio is 1.0, the relative cholestanol-to-cholesterol ratio varied from 1.0 in plasma and liver to 30.0 in the cerebellar xanthoma; cholestanol was especially high in nerve tissue. Our data indicate that CTX patients absorb cholestanol from the diet. They have a higher than normal cholestanol production rate. Cholestanol was derived from cholesterol. In CTX patients, the blood-brain barrier was intact to the passage of [4-14C]cholesterol and [4-14C]cholestanol. The deposition of large amounts of cholestanol (up to 30% of total sterols in cerebellum) in nerve tissues must have an important role in the neurological symptoms in CTX patients. In view of the intact blood-brain barrier, several other explanations for the large amounts of cholestanol in the brain were postulated.
Supplementary key words neurological disorders • cholesterol and cholestanol turnover • blood brain barrier • brain sterols • brain cholestanol • chenodeoxycholic acid • sterol 27-hydroxylase • 27-hydoxy cholesterol • 7
-hydroxy-4 cholesten-3-one
Cerebrotendinous xanthomatosis (CTX), first reported by van Bogaert, Scherer, and Epstein (1) in 1937, is a rare autosomal recessive neurologic disease characterized by xanthomas of tendons, lungs, and the brain despite normal or low plasma cholesterol concentrations. Other major clinical manifestations include diarrhea, cataracts, premature atherosclerosis and myocardial infarction, progressive cerebellar ataxia, dementia, spinal cord paresis, and below-normal intelligence. Greatly increased concentrations of cholestanol, the 5-dihydro derivative of cholesterol, in plasma and in practically all tissues, notably xanthoma, nerve tissues, and brain, have been reported (2–4).
Small amounts of cholestanol normally accompany cholesterol in virtually every mammalian tissue. The normal biosynthesis and metabolism of cholestanol have been well reviewed by Bjorkhem and Skrede (4). The major metabolic defect in the CTX syndrome is the impaired synthesis of chenodeoxycholic acid from cholesterol. There is a deficiency of the mitochondrial sterol 27-hydroxylase enzyme, which catalyzes the initial steps in the oxidation of the side chain of the cholesterol structure in the conversion of cholesterol to bile acids (3–6). The CTX syndrome has been traced to a mutation of the sterol 27 hydroxylase (CYP27A1) gene on chromosome 2q33-qter (7). Several other mutations of the sterol 27-hydroxylase gene have now been reported (7–11). It has been suggested that the accumulation of cholestanol in the tissues is secondary to this mutation of the CYP27A1 gene (3).
Salen, Shefer, and Berginer (3) proposed that large accumulations of cholesterol and cholestanol in tissues result from an increased synthesis of cholestanol and cholesterol. Besides increased biosynthesis of cholestanol, increased intestinal absorption of dietary cholestanol may also contribute to the plasma levels, so we studied the intestinal absorption of cholestanol in two patients with CTX. We also measured the turnovers of plasma cholestanol and cholesterol and determined the equilibration of isotopic cholestanol and cholesterol between plasma, xanthoma, and peripheral nerve tissue. In addition, we also analyzed 14 autopsy samples from one patient to assess the cholestanol and cholesterol contents and their relative ratios in various tissues. In particular, we suggest some new hypotheses for the profound accumulation of cholestanol in nerve tissue, which may result from synthesis in nerve tissue.
MATERIALS AND METHODS
Patients
Two brothers, aged 26 and 30 years and weighing 78 and 68 kg, respectively, displayed all of the clinical manifestations of the CTX syndrome: large tendon xanthoma (Achilles, patellar, and extensor tendons of the hands), cataracts, ataxia, and below-normal intelligence. Their plasma cholestanol concentrations were 3.5 and 3.6 mg/dl, respectively, and plasma cholesterol levels were 195 and 181 mg/dl, respectively. The plasma cholestanol concentrations of their mother, eldest brother and his two children, and a maternal uncle were normal (0.25–0.45 mg/dl).
Both patients were studied at the Clinical Research Center of the University of Iowa Hospital in the 1970s. The experimental protocols were explained to the patients, their mother, and their court-appointed protector, and informed consent was obtained from all persons concerned according to the policies of the Committee on Investigation involving Human Beings of the University of Iowa College of Medicine. The Committee approved the study protocol. At that time, their treatment was a low-cholestanol diet; the value of chenodeoxycholic acid in the CTX syndrome had not yet been demonstrated.
Cholestanol absorption
The intestinal absorption of cholestanol was measured in both patients by feeding a single dose of [4-14C]cholestanol in a formula breakfast that provided 800 calories and contained 50 g of fat, consisting of 19% monounsaturated, 16% polyunsaturated, and 18% saturated fatty acids. The carbohydrate and protein contents of the meal were 63 and 25 g, respectively. The cholesterol content was 100 mg. The isotope (3 µCi) along with 2.5 µCi of [22,23-3H]ß-sitosterol was dissolved in 5 g of peanut oil with 150 mg of crystalline ß-sitosterol and 0.5 mg of crystalline cholesterol added as carriers and then mixed with egg yolk. An aliquot of the formula was analyzed for sterol mass and radioactivities to obtain the exact amounts fed (12). Feces were collected daily for the next 7 days and stored in plastic bags at –20°C. The 7 day feces pool was homogenized with water (1:1), and an aliquot was analyzed for radioactivity in the neutral sterol fraction (12). The absorption of cholestanol was calculated as the difference between the amount fed and the amount excreted in the neutral sterol fraction of the feces after correcting on the basis of sitosterol radioactivity recovery and was expressed as percentage of intake (13).
Cholesterol and cholestanol turnover
For the cholesterol turnover studies, both patients were given intravenously a single dose of 50 µCi of [4-14C]cholesterol (New England Nuclear Corp., Boston, MA) dissolved in 5 ml of ethanol and suspended in 500 ml of 0.9% NaCl solution as described previously (14). A plasma cholestanol turnover study was carried out in both patients 2 years later. Approximately 50 µCi of [4-14C]cholestanol [New England Nuclear; purified by AgNO3-TLC, with>99% radiopurity as checked by AgNO3-TLC and gas liquid chromatography (GLC)] was given intravenously as a single dose as described above.
Venous blood samples were obtained every morning in the fasting state for the first 5 days and then twice weekly for the next 12 weeks. Plasma was separated by centrifugation at 4°C and stored at –20°C for determination of plasma cholestanol- and cholesterol-specific radioactivities as described below.
Plasma cholestanol- and cholesterol-specific radioactivities were plotted semilogarithmically against time. For both patients, the plasma cholesterol-specific radioactivity decay curve could be resolved precisely into the sum of two exponentials. The various parameters of cholesterol turnover were calculated on the basis of the kinetic analysis of the two-pool model (15). However, the semilogarithmic plots of the plasma cholestanol-specific radioactivity were widely scattered in both patients. In patient 1, the plasma cholestanol-specific radioactivity decay curve could be resolved, although not very precisely, into the sum of two exponentials; therefore, approximate values for cholestanol turnover were calculated. In patient 2, the plasma cholestanol-specific radioactivity decay curve could not be resolved into the sum of two exponentials. Hence, no attempt was made to calculate the plasma cholestanol turnover in this patient.
Biopsies of tendon xanthomas, sural nerve, liver, skin, and adipose tissue were obtained using routine hospital procedures. Duodenal bile samples were obtained by duodenal intubation using MgSO4 solution to enhance the bile flow. All procedures were carried out while both patients were inpatients at the Clinical Research Center.
Determination of sterol concentrations in plasma, erythrocytes, and tissues
One milliliter of plasma or serum was saponified with alcoholic KOH and extracted with hexane according to the procedure of Abell et al. (16). The erythrocytes were washed repeatedly with 0.9% NaCl solution, and the lipids were extracted with chloroform-isopropanol (7:11, v/v) according to the method of Rose and Oklander (17) and made up to 50 ml with the solvent. All tissue specimens were cut into small pieces and washed repeatedly with 0.9% NaCl solution to remove blood. The pieces were blotted on a filter paper, weighed, and dried in a hot vacuum oven at 80°C to a constant weight. The dried tissue was repeatedly extracted by boiling with chloroform-methanol (2:1, v/v); the lipid extract was made up to a suitable volume (usually 50 ml) with the solvent. An aliquot was then saponified and extracted with hexane as described above. The hexane extract was evaporated to dryness under N2, redissolved in a small volume of hexane, and subjected to AgNO3-TLC using a 0.25 mm thick silica gel H-coated glass plate (Applied Science Laboratories, Inc., State College, PA). The plate was pre-run in chloroform, air-dried, and impregnated with AgNO3 by allowing a 10% aqueous solution of AgNO3 to migrate to the top. The plate was then air-dried and activated at 120°C for 1 h. The hexane extract was applied on the plate as a narrow band using the TLC streaker (Applied Science Laboratories) along with reference cholestanol and cholesterol. The plate was developed at 4°C in chloroform-acetone (97:3, v/v), and the solvent was allowed to ascend to the top of the plate. The sterol bands were visualized under ultraviolet light after spraying lightly with a saturated aqueous solution of Rhodamine 6G (Applied Science Laboratories), scraped, eluted with ethyl ether, and subjected to GLC as the trifluoroacetate derivative, with 5-cholestane as the internal standard (18).
GLC was carried out on a dual-column gas chromatograph equipped with a hydrogen flame ionization detector and an automatic digital integrator (Hewlett-Packard Co., Avondale, PA). The column was a coiled glass of 183 cm length and 2 mm internal diameter packed with 3% QF-1 on 80–100 mesh Gas-chrome Q (Supelco, Inc., Bellefonte, PA). The temperatures of the column, detector, and flash heater were 210, 240, and 260°C, respectively. Nitrogen was the carrier gas at a flow rate of 24 ml/min, and the inlet pressure was 50 p.s.i. The sensitivity of our GLC method for the detection of sterols was in the 10–20 ng range.
Free and esterified sterols in the tissue extracts were separated by TLC using a silica gel G plate. The plate was developed in petroleum ether-diethyl ether-glacial acetic acid (80:20:1, v/v). The free and ester sterol bands were scraped, eluted with diethyl ether, and subjected to AgNO3-TLC as described above to separate cholestanol from cholesterol. The individual sterols were quantitated by GLC as described above.
Determination of cholestanol and cholesterol radioactivities
Cholestanol and cholesterol radioactivities in the plasma and tissues were determined on aliquots of the same eluate obtained after separation of cholestanol from cholesterol by AgNO3-TLC. Ten milliliters of scintillation fluid [4 g of 2,5,5-diphenyloxazole and 0.1 g of l,4-bis(2-5-phenyloxazolyl) benzene per liter of toluene] was used in each counting vial. A liquid scintillation spectrometer equipped with external standardization (Packard Instrument Co., Inc., Downers Grove, IL) was used for radioactivity counting.
RESULTS
Plasma lipid concentrations
The plasma levels of cholestanol, cholesterol, and triglyceride as well as erythrocyte concentrations of cholestanol and cholesterol in the two patients are shown in Table 1
. Both patients had more than eight times higher concentrations of plasma cholestanol than for the normal subjects. Cholestanol was also found in the membranes of erythrocytes. The ratios of cholestanol to cholesterol in the plasma and erythrocytes were similar, showing ready equilibration. Plasma cholesterol levels were <200 mg/dl in both patients. Plasma triglyceride levels were normal.
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39 mg/day (Table 3).
Tissue sterols, radioactivity, and the blood-brain barrier
Cholestanol was found in large amounts in tendon xanthoma, peripheral nerve, skin, and adipose tissue of patient 1 (Table 4
). It constituted between 6% and 12% of total sterols in the tendon xanthomas, skin, and adipose tissue. In the sural nerve, cholestanol constituted 20% of total sterols. In patient 2, in skin and adipose tissue, cholestanol constituted 7% and 18% of total sterols, respectively. The relative cholestanol-cholesterol ratios varied from 1.0 in plasma to 13.5 in sural nerve in patient 1. It varied from 1.0 in plasma to 11.0 in adipose tissue in patient 2.
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The free and ester cholesterol-specific radioactivities of the left tendo-Achilles xanthoma biopsied 49 days after intravenous administration of [4-14C]cholesterol were 240 and 184 dpm/mg cholesterol, and those for xanthoma cholestanol were 228 and 140 dpm/mg for free and ester cholestanol, respectively (Table 5 ). The plasma free and ester cholesterol-specific radioactivities on the day of biopsy were 247 and 251 dpm/mg cholesterol. Similarly, free and ester cholesterol-specific activities of the right tendo-Achilles xanthoma biopsied 87 days after injection of [14C]cholesterol were 252 and 149 dpm/mg cholesterol, respectively, whereas plasma cholesterol-specific activities were 152 and 151 dpm/mg and those for cholestanol were 166 and 111 dpm/mg. Furthermore, free and esterified cholesterol-specific activities in the left patellar xanthoma obtained 415 days after intravenous [14C]cholesterol were 165 and 58 dpm/mg, and those for cholestanol were 105 and 34 dpm/mg. In the plasma, no radioactivity was detected at 415 days.
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Isotopic equilibration between liver, bile, and plasma cholestanol after intravenous [4-14C]cholestanol
The data presented in Table 6
show that liver cholestanol-specific activity was more than double the plasma cholestanol-specific activity in both patients at 7–8 days after an intravenous dose of [4-14C]cholestanol. Biliary cholestanol-specific activity equilibrated to only 22% of that in liver in patient 1 and to 47% in patient 2.
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The principal findings of this investigation are as follows: 1) the blood-brain barrier was intact to the passage of both cholesterol and cholestanol; 2) there was an especially high content of cholestanol in the nerve tissue, which led to an especially high cholestanol-to-cholesterol ratio (the implication of these findings is that brain synthesis accounts for the large amount of cholestanol in the brain); 3) the whole body synthesis of cholestanol was also increased; and 4) there was cholestanol absorption from the diet. Each of these topics will be discussed sequentially.
The peripheral nerve biopsies obtained at 49 and 87 days after an intravenous dose of [4-14C]cholesterol did not show any radioactivity in either the cholesterol or the cholestanol fraction of the nerve. This finding suggested that the brain-nerve barrier for these sterols was intact in these patients. This barrier would prevent the transport of sterols from the blood into nerve tissue. The blood-nerve barrier is similar to the blood-brain barrier (19). The blood-brain barrier, therefore, is intact in these patients as well. Our finding of a high cholestanol-to-cholesterol ratio in the nerve tissue, compared with liver and other tissues, is consistent with this interpretation. Furthermore, with a disrupted blood-brain barrier, plant sterols, such as sitosterol, should be found in milligram quantities in the brain. We found no plant sterols. Three other analyses of brain tissue from CTX patients, by Menkes, Schimschock, and Swanson (20), Salen (21), and Philippart and van Bogaert (22), did not allude to plant sterols in their GLC analyses. These data further attest to the intactness of the blood-brain barrier in the CTX syndrome. However, Salen and colleagues (23) have postulated that bile acid alcohols in CTX alter the blood-brain barrier and allow circulating apolipoprotein B-100 to enter the central nervous system. The reason for this discrepancy is not clear.
The two CTX brothers whom we studied had already suffered severe neurological damage. We surmise that this disease is progressive with time. The older the patient, the greater the deposition of cholestanol in the nerve tissue and the greater the ratio of cholestanol to cholesterol in the brain. This progression is also seen in the cataract formation in the lens, which does not become manifested until late childhood. CTX children are probably normal at birth but may develop intractable diarrhea. This "cholestanol progression" of the disease makes therapy with chenodeoxycholic acid even more important (6), and early diagnosis by genetic screening at birth would be important to implement. However, still to be understood is how therapy with chenodeoxycholic acid will stop cholestanol production in the brain. The decline of cholestanol levels in cerebrospinal fluid (23) suggests that chenodeoxycholic acid crosses the blood-brain barrier and, as a product, then inhibits cholestanol production in the brain, liver, and other tissues by feedback inhibition.
Norlin and colleagues (24) have postulated a number of pathways in the CTX syndrome that can lead to increased amounts of cholestanol in the tissues. All tissues of the body express the CYP27A1 gene, so the metabolism of oxysterols may be important in extrahepatic tissues as well as in the liver. An attractive mechanism is that in CTX, the accumulation of cholestanol could be caused by a reduced removal of the sterol from macrophages because of a deficiency in CYP27A1 in the macrophages (6). Menkes, Schimschock, and Swanson (20), the discoverers of cholestanol storage in this disease, had, in fact, postulated a defect in the transport of cholesterol across the cell membrane in CTX syndrome, with the result that excess cholesterol may be converted to cholestanol, which is stored either as the free or the esterified form. This hypothesis has, as one of its attractive premises, that there is a marked limitation of capacity for the removal of cholestanol from cells. The data of Gardner-Medwin et al. (25) and Derby et al. (26) on the in vivo labeling of cerebral and cerebellar sterols in one CTX patient provide some support to the idea of a deficiency in the mechanism for the removal of cholestanol from tissues. Bjorkhem (27) showed net flux of 7-hydroxy-3-oxo-4 cholestenoic acid out of the human central nervous system. Because this oxysterol is a metabolic product of 27-hydroxycholesterol, it is conceivable that a reduction in output of this oxysterol could lead to cholestanol overaccumulation in the brain. We suggest that HDL, because of its well-known role in the reverse cholesterol transport process, may be involved. In CTX patients, HDL cholesterol and apolipoprotein A composition have been reported to be abnormally low (28).
However, the most attractive explanation for a large amount of cholestanol in the tissues of CTX patients is an exaggerated synthesis of cholestanol as a result of the C27-hydroxylase deficiency (6). The synthesis of cholestanol has been depicted in elegant pathways by Norlin and colleagues (24). Perhaps the simplest pathway is that the 7-hydroxy-4-cholesten-3-one formed from cholesterol in the tissues is the normal precursor for chenodeoxycholic acid. In the absence of CYP27A1, chenodeoxycholic acid is not formed, and the secondary pathway to cholestanol from 7-hydroxy-4-cholesten-3-one results. This possible alternative pathway for the accumulation of cholestanol in the brain of CTX patients could result from the uptake of this bile acid precursor (e.g., 7-hydroxy-4-cholesten-3-one) from the blood by the brain. This compound is known to be converted into cholestanol in peripheral tissues (29). But how does the explanation for the hepatic synthesis of cholestanol apply to the nervous system, where until now bile acid synthesis has not been demonstrated? Recently, Mano and colleagues (30) showed that chenodeoxycholic acid is synthesized from 3ß-hydroxy-5-cholenoic acid by rat brain enzyme systems, and Nariyasu et al. (31) detected bile acids in the rat brain. This cholenoic acid may be synthesized from cholesterol via 24-hydroxy cholesterol as an intermediate (30). Large amounts of 24-hydroxy cholesterol exist in rat brain microsomal fractions (32). Although there is no evidence that chenodeoxycholic acid can be synthesized from cholesterol in the brain, we speculate that there may be a pathway from 24-hydroxy cholesterol to bile acid in the brain. The mutation of the CYP27A1 gene (identified in the brain) may conceivably lead to disturbances in the synthesis of chenodeoxycholic acid, so that small, daily amounts of cholestanol are produced in the brain over many years. This explanation would help account for the large amount of cholestanol in the central and peripheral nervous systems.
One other defect in cholestanol metabolism in the CTX syndrome was reported to be the increased production of cholestanol in the body. Salen and Grundy (33) showed that cholestanol turnover in two CTX patients and in five human subjects (one normal and four hyperlipidemic) conformed to the two-pool kinetics described by Goodman and Noble (15). They reported that PRA values in two CTX patients were 48 and 57 mg/day (average, 52.5 mg/day), much greater than those in the control subjects (mean, 11.8 ± 6.0 mg/day; range, 6–18 mg/day). The production rate of cholestanol in our patient was 39 mg/day, lower than the average value reported by Salen and Grundy (33) in their two patients; yet, our value is 3.5 times higher than the mean value of 11.8 mg/day reported for five normal human subjects by those authors. Thus, we concur with the conclusion of Salen and Grundy (33) that in the CTX syndrome, the biosynthesis of cholestanol for the whole body is increased.
In our two CTX patients, the intestinal absorption of cholestanol was found to be 5.5% and 3.4% after a single oral test meal containing [4-14C]cholestanol (Table 2). In six normal human subjects, the intestinal absorption of cholestanol was 3.3 ± 2.7% of the dose. In the1930s, using balance methods, Burger and Wintersteel (34) and Dam (35) could not demonstrate cholestanol absorption in normal humans. To the best of our knowledge, no other studies on cholestanol absorption in CTX have been reported. Thus, the capacity to absorb cholestanol from the diet by CTX patients has implications for the dietary therapy of this disease, because cholestanol is present in certain foods, such as eggs and high-fat dairy products.
The great accumulation of cholestanol in the cerebrum, cerebellum, and peripheral nerves suggests that cholestanol in all probability plays a key role in the neurological dysfunction of CTX patients. Structurally, cholestanol is a saturated sterol compared with the unsaturated cholesterol, and it may have different electrical conductivity than cholesterol. Replacing large amounts of cholesterol with cholestanol (up to 30%) in the nerve tissue conceivably would have significant effects upon function. Philippart and van Bogaert (22) suggested that a part of the cholesterol normally present in myelin as the integral part of the cell membrane structure is replaced by cholestanol, thereby altering the cellular membrane structure and producing the neurological dysfunction observed in CTX. Stahl, Sumi, and Swanson (36) have demonstrated the presence of free cholestanol in myelin. The change in myelin lipid composition has also been observed in the frontal lobe of the cerebrum. This change in myelin composition may account for the severe changes in mentation that are so typical in CTX.
In conclusion, although the underlying defect(s) producing the clinical manifestations in the CTX syndrome, particularly the neurological manifestations, has yet to be established, the presence of high concentrations of cholestanol in relation to cholesterol in the brain may have important pathological consequences in CTX patients. Several hypotheses for the increased cholestanol concentration in the brain of these patients have been postulated based upon the presence of CYP27A1 and the synthesis of chenodeoxycholic acid by a brain enzyme system. In the absence of the CYP27A1 gene, the pathway to chenodeoxycholic acid from cholesterol is blocked, with diversion then to the synthesis of cholestanol. This hypothesis fits the data of an intact blood-brain barrier for cholestanol and cholesterol and the high cholestanol-to-cholesterol ratio in the brain compared with the plasma and other nonnerve tissue. Cholestanol may be synthesized in the brain from cholesterol or from a circulating precursor to chenodeoxycholic acid synthesis that enters the brain. The removal of cholestanol from the brain could also be impaired and result in its accumulation in nerve tissue.
ACKNOWLEDGMENTS
This study was supported by research Grants HE-l4230 and HL-08974 from the National Heart, Lung, and Blood Institute and from the General Clinical Research Centers Program (Grants MOI-FR-59 and 5 M01 RR-000334) of the Division of Research Resources, National Institutes of Health (Bethesda, MD). The expert technical assistance of Mary J. Garst, Susan Weickert, and Adrianne Hakes is gratefully acknowledged. The authors are grateful to Dr. William H. Elliot (Department of Biochemistry, St. Louis University School of Medicine) for kindly providing a pure sample of allocholic acid (3,7,l2-trihydroxy-5-cholan-24-oic acid). The authors thank Dr. Anuradha Pappu of our department for helping in the discussion of bile acids and cholestanol and Carolyn Reid for the careful preparation of many revisions of the manuscript. Finally, the authors are grateful for the participation of the two brothers with CTX in our studies and to their mother, who encouraged them. Some of these studies were carried out when the investigators were affiliated with the General Clinical Research Center and the Department of Medicine, University of Iowa College of Medicine (Iowa City, IA).
Manuscript received March 7, 2006 and in revised form April 20, 2006 and in re-revised form June 22, 2006 and in re-re-revised form August 2, 2006. and in re-re-re-revised form September 12, 2006. and in re-re-re-re-revised form September 29, 2006.
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