The natural history of phytosterolemia: Observations on its homeostasis.

BACKGROUND AND AIMS: Phytosterolemia is a rare genetic disease caused by mutation of the ABCG5/8 gene. Our aim was to elucidate the natural history and homeostasis of phytosterolemia. METHODS: We analyzed a Hutterite kindred consisting of 21 homozygotes with phytosterolemia assembled over a period of two decades, all of whom carried the ABCG8 S107X mutation and were treated with ezetimibe. RESULTS: Most of these subjects were asymptomatic and devoid of clinical stigmata, and this, since they were ascertained primarily by a process of cascade testing, suggests that, relative to its true prevalence, phytosterolemia is a condition of low morbidity. All subjects have responded well to treatment with ezetimibe. Initial (pre-treatment) and post-ezetimibe levels of cholesterol and sitosterol were measured and percentage changes on ezetimibe were calculated. We found initial levels to be inversely related to subjects' ages as were percentage responses to ezetimibe therapy. There was also a direct correlation between initial levels and percentage responses to ezetimibe. Hence on-treatment levels were very uniform. CONCLUSIONS: This evidence of a link with age leads us to propose that an age-related change in cholesterol and sterol homeostasis occurs at puberty in phytosterolemia and that the change is due to high sterol and/or stanol levels causing feedback inhibition of sterol regulatory element-binding protein (SREBP-2) processing. This would explain the well-documented phenomenon of depressed cholesterol synthesis in phytosterolemia. It is also well-known that LDL-receptor activity is increased, and this feasibly explains reduced LDL levels and consequent reduction of plasma cholesterol and sitosterol levels. Downregulated SREBP-2 processing would be expected to also lower proprotein convertase subtilisin/kexin type 9 (PCSK9) levels and this would explain high LDL-receptor activity. The above state could be termed disrupted homeostasis and the alternative, seen mostly in children and characterized by hypercholesterolemia and hypersterolemia, simple homeostasis.

Mifepristone treatment results in differential regulation of glycerolipid biosynthesis in baby hamster kidney cells expressing a mifepristone-inducible ABCA1.

ATP binding cassette A1 (ABCA1) transports cholesterol, phospholipids and lipophilic molecules to and across cellular membranes. We examined if ABCA1 expression altered cellular de novo glycerolipid biosynthesis in growing Baby hamster kidney (BHK) cells. Mock BHK cells or cells expressing a mifepristone-inducible ABCA1 (ABCA1) were incubated plus or minus mifepristone and then with [(3)H]serine or [(3)H]inositol or [(3)H]ethanolamine or [methyl-(3)H]choline or [(3)H]glycerol or [(14)C]oleate and radioactivity incorporated into glycerolipids determined. Mifepristone did not affect [1,3-(3)H]glycerol or [(14)C]oleate or [(3)H]ethanolamine or [methyl-(3)H]choline uptake in BHK cells. In contrast, [(3)H]glycerol and [(14)C]oleate incorporated into phosphatidylserine (PtdSer) were elevated 2.4-fold (p < 0.05) and 54% (p < 0.05), respectively, upon ABCA1 induction confirming increased PtdSer biosynthesis from these precursors. However, mifepristone inhibited [(3)H]serine uptake and incorporation into PtdSer indicating that PtdSer synthesis from serine in BHK cells is dependent on serine uptake. Mifepristone stimulated [(3)H]inositol uptake in mock and ABCA1 cells but not its incorporation into phosphatidylinositol indicating that its synthesis from inositol is independent of inositol uptake in BHK cells. [(3)H]glycerol and [(14)C]oleate incorporated into triacylglycerol were reduced and into diacylglycerol elevated only in mifepristone-induced ABCA1 expressing cells due to a decrease in diacylglycerol acyltransferase-1 (DGAT-1) activity. The presence of trichostatin A, a class I and II histone deacetylase inhibitor, reversed the ABCA1-mediated reduction in DGAT-1 activity but did not affect DGAT-1 mRNA expression. Thus, mifepristone has diverse effects on de novo glycerolipid synthesis. We suggest that caution should be exercised when using mifepristone-inducible systems for studies of glycerolipid metabolism in cells expressing glucocorticoid responsive receptors.

Quantification of serum apolipoprotein B by infrared spectroscopy.

While the conventional approach to assessing both the risk of coronary artery disease and the adequacy of therapy is LDL cholesterol testing, there is compelling evidence to suggest that apolipoprotein B (apoB) is superior to LDL cholesterol for both of these purposes. However, the measurement of apoB requires techniques that can be expensive and difficult to standardize. The aim of this study was, therefore, to develop a new method, based on infrared (IR) spectroscopy, for the routine quantification of apoB in human serum. A total of 366 serum samples were obtained from patients with various disorders. Small volumes (2 microl) of serum specimens were dried to films, and duplicate IR absorption spectra measured. The reference apoB concentrations were determined separately using a standard method, and the proposed IR method was then calibrated using partial least squares (PLS) regression analysis to quantitatively correlate the IR spectra with the reference results. The apoB concentrations predicted from the IR spectra of serum were highly correlated and in excellent agreement with those determined by the reference method. The correlation coefficient (r) for apoB was 0.94, with the standard error between IR-predicted and reference values was 0.10 g/L. In combination with earlier work demonstrating the accurate determination of LDL-C, HDL-C, total cholesterol, and triglycerides from a single infrared spectroscopic measurement, the addition of accurate apoB determination from the same spectrum makes the method very attractive for laboratory use in the routine evaluation of coronary artery disease risk.

Apolipoprotein(a) phenotypes predict the severity of coronary artery stenosis.

BACKGROUND: Studies on the impact of elevated levels of lipoprotein(a) (Lp[a]) or apolipoprotein(a) (apo[a]) on the development of coronary artery disease have given controversial results. The relationship between apo(a) phenotypes and coronary artery stenosis remains unclear. METHODS: Lipid profiles, and apo(a) levels and phenotypes were analyzed in 225 patients who underwent elective coronary angiography. Coronary artery stenosis, as indicated by angiography, was estimated by a newly devised minimal lesion (ML) grading system. Relationships between lipoprotein variables and coronary artery stenosis were examined by linear and logistic regression models. RESULTS: On the basis of ML score, patients with larger apo(a) phenotypes (S3, S3a or S4) had a lower rate of coronary artery stenosis (68%-76%) than those with smaller phenotypes (S1, S1a, S2 or S2a - 79%-95%). The odds of coronary artery stenosis in patients with smaller apo(a) phenotypes were significantly different from those of patients with larger phenotypes (p < 0.001). Also, patients with a history of myocardial infarction, angina, hypertension, diabetes or hypercholesterolemia were more likely to show coronary artery stenosis on angiography. With respect to lipid levels, 20.2% of patients had an elevated serum total cholesterol (TC) level and 16.1% an elevated low-density lipoprotein cholesterol (LDL-c) level. In 21.3%, the high-density lipoprotein cholesterol (HDL-c) level was decreased. There were significant positive correlations of serum TC with those of the TC/HDL-c ratio, LDL-c, triglycerides and HDL-c (p < 0.05 and 0.001), of LDL-c with TC and apo(a) (p < 0.001) and of ML scores with the TC/HDL-c ratio and patient age (p < 0.01 and 0.001). There were significant negative correlations of TC and apo(a) levels with apo(a) phenotypes (p < 0.05 and 0.001) and of ML scores with HDL-c (p < 0.001). The odds of coronary artery stenosis in patients with abnormally high apo(a) levels (44.6%) were not significantly different from those of patients with apo(a) levels in the normal range. INTERPRETATION: Smaller apo(a) phenotypes, but not elevated levels of apo(a), may help to predict the rate and severity of coronary artery stenosis. HDL-c independently and negatively correlated with the extent of the stenosis.

Reagent-free, simultaneous determination of serum cholesterol in HDL and LDL by infrared spectroscopy.

BACKGROUND: The purpose of this study was to assess the feasibility of infrared (IR) spectroscopy for the simultaneous quantification of serum LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) concentrations. METHODS: Serum samples (n = 90) were obtained. Duplicate aliquots (5 microL) of the serum specimens were dried onto IR-transparent barium fluoride substrates, and transmission IR spectra were measured for the dry films. In parallel, the HDL-C and LDL-C concentrations were determined separately for each specimen by standard methods (the Friedewald formula for LDL-C and an automated homogeneous HDL-C assay). The proposed IR method was then developed with a partial least-squares (PLS) regression analysis to quantitatively correlate IR spectral features with the clinical analytical results for 60 randomly chosen specimens. The resulting quantification methods were then validated with the remaining 30 specimens. The PLS model for LDL-C used two spectral ranges (1700-1800 and 2800-3000 cm(-1)) and eight PLS factors, whereas the PLS model for HDL-C used three spectral ranges (800-1500, 1700-1800, and 2800-3500 cm(-1)) with six factors. RESULTS: For the 60 specimens used to train the IR-based method, the SE between IR-predicted values and the clinical laboratory assays was 0.22 mmol/L for LDL-C and 0.15 mmol/L for HDL-C (r = 0.98 for LDL-C; r = 0.91 for HDL-C). The corresponding SEs for the test spectra were 0.34 mmol/L (r = 0.96) and 0.26 mmol/L (r = 0.82) for LDL-C and HDL-C, respectively. The precision for the IR-based assays was estimated by the SD of duplicate measurements to be 0.11 mmol/L (LDL-C) and 0.09 mmol/L (HDL-C). CONCLUSIONS: IR spectroscopy has the potential to become the clinical method of choice for quick and simultaneous determinations of LDL-C and HDL-C.