High resolution-magic angle spinning (HR-MAS) NMR-based metabolomic fingerprinting of early and recurrent hepatocellular carcinoma

In order to elucidate the metabolite changes associated with hepatocellular carcinoma (HCC) oncogenesis and progression, we compared the profiles obtained by 1D proton HRMAS NMR spectroscopy of 51 needle biopsies (14 primary nodules, 14 recurrent, and 23 paired cirrhotic specimens). The diagnosis of HCC was based on 2 concordant imaging techniques and was confirmed by histology in 20 cases. Spectroscopy was performed using a Bruker AVANCE II 600 spectrometer. One-dimensional proton spectra were acquired using water-suppressed (noesygppr) pulse and spin-echo CPMG sequences. Signals were assigned by BBIOREFCODE and were confirmed by HSQC. Statistics was based on the SIMCA P package. Orthogonal projection to latent structure (OPLS-DA) showed a clear separation between tumor and cirrhosis. This difference was maintained when the analysis of paired samples from primary to recurrent nodules was split. OPLS-DA of primary and recurrent nodules also showed a significant difference. The relationship between metabolite profile and HCC volume was evaluated comparing the spectra obtained in tumors ≤2 cm (n = 15) and in those larger than 2 cm (n = 11). Univariate comparison of the most relevant metabolites showed that: (1) increased choline, TMAO, and decreased saturated fatty acids differentiate HCC from the surrounding tissue; (2) increased lactate and myo-inositol differentiate recurrent from primary HCC; (3) decreased saturated fatty acids characterize large HCC nodules.     

In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity

Previous studies have established that human hepatocellular carcinoma cells (Hep G2) secrete into serum-free medium the pro form of apolipoprotein A-I (proapo-A-I) suggesting that its conversion to mature apo-A-I occurs after secretion. In order to assess the mode and site of proapo-A-I to apo-A-I conversion, we incubated the medium from [3H]proline-labeled Hep G2 cells with either human plasma, serum, lymph, or fractions thereof obtained by density gradient ultracentrifugation. The conversion was monitored by two-dimensional gel electrophoresis and by Edman degradation. Human plasma, serum, or mesenteric lymph all induced proapo-A-I to apo-A-I conversion; this was time dependent, unaffected by the serine protease inhibitor phenylmethylsulfonyl fluoride and inhibited by EDTA. Purified radiolabeled proapo-A-I bound to lymph chylomicrons and plasma high density lipoproteins. The converting enzyme was associated with both of these particles. Activity was also found in the d greater than 1.21-g/ml fraction and may have been derived from high density lipoprotein after displacement by high salts and/or ultracentrifugal force. We conclude that the conversion of proapo-A-I to apo-A-I occurs extracellularly and is probably effected by a metallo-enzyme which may act at the amphiphilic surface of either chylomicrons or high density lipoproteins.     

Human proapolipoprotein A-II is cleaved following secretion from Hep G2 cells by a thiol protease

The two principal high-density lipoprotein apolipoproteins A-I and A-II are both initially synthesized as preproproteins. The prosegment of apo-A-I is unusual: it ends with paired glutamine residues and is removed extracellularly. The apo-A-II prosegment resembles the propeptides of prohormones and proalbumin: it ends with paired basic amino acids. We have studied the processing of proapo-A-II in a human hepatoma cell line (Hep G2) which is known to accurately and efficiently remove the prosegment from proalbumin prior to secretion. Pulse-chase experiments were performed in order to determine if the apo-A-II prosegment is removed prior to or after secretion. Apo-A-II was purified from cell lysates and media at various times during the chase and subjected to automated sequential Edman degradation. The results indicate that proteolytic processing of proapo-A-II is largely an extracellular event. These cells secrete the protease responsible for prosegment removal. The converting activity present in media is not blocked by serine protease inhibitors (phenylmethanesulfonyl fluoride, aprotinin, and furoyl saccharin) or by a metalloprotease inhibitor (o-phenanthroline). It is inhibited by the thiol protease reagents p-chloromercuribenezene-sulfonic acid and leupeptin. Prosegment removal changes the pI of the dominant apo-A-II isoform from 6.61 to 4.95. The presence of the propeptide does not prevent specific in vitro recombination of apo-A-II with high-density lipoprotein3 particles present in normolipemic serum. Extracellular processing after a single basic amino acid has been described for a variety of precursor proteins. Extracellular cleavage of the apo-A-II propeptide after paired COOH-terminal basic residues represents a novel processing pathway.     

Apolipoprotein B-100 of plasma low density lipoproteins undergoes proteolysis by contact activation factors when plasma is treated with dextran sulfate-500-MgCl2

Human plasma low density lipoproteins (LDL) isolated by ultracentrifugation showed a single band corresponding to apolipoprotein B-100 (apoB-100) by SDS-gradient gel electrophoresis (GGE). In turn, apoB-100 of LDL precipitated from plasma by dextran sulfate-500 (DS)-MgCl2 exhibited several bands indicative of a degradative process. The degradation was more extensive at 0 degrees C than at either 23 degrees C or 37 degrees C, and appeared to be related to a protease activity that cleaved both the synthetic peptide, Z-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) and apoB-100. Proteolysis was proportional to the DS added to the plasma, was prevented by the kallikrein inhibitor, D-Phe-L-Phe-L-Arg-CHCl2, and was significantly decreased in plasma specimens of patients with either factor XII or prekalikrein deficiency. LDL pre-purified by ultracentrifugation and then precipitated by DS in the absence of plasma exhibited no proteolysis. However, proteolysis was observed when LDL interacted with kallikrein. The two main apolipoproteins of HDL3, apoA-I and apoA-II, were not affected by this proteolytic process. We interpret the results to indicate that the negatively charged surface provided by DS accelerates in plasma the autoactivation of factor XII and the activation of prekallikrein, resulting in an increase of the effective concentration of kallikrein and possibly other proteases and proteolysis of LDL-apoB-100. The higher degree of the DS-induced proteolysis of apoB-100 at 0 degrees C than at 23 degrees C is likely the consequence of enhanced autoactivation of factor XII and a decreased efficiency of plasma inhibitors, such as C1-inhibitor. We speculate that the proteolysis of apoB-100 induced by DS is not limited to this polyanion, but may also be the property of other negatively charged agents, particularly at cold temperatures.     

Study of abnormal plasma low-density lipoprotein in rhesus monkeys with diet-induced hyperlipidemia.

Male rhesus monkeys were divided into three groups: five were fed a regular primate chow diet and were used as controls; four received an "average" American diet; and five a special low-fat primate chow diet supplemented with 25% coconut oil and 2% cholesterol. In all of these animals, the plasma low-density lipoproteins (LDL) were isolated by ultracentrifugal flotation between densities of 1.019 and 1.050 g/ml. The LDL of the five control monkeys had variable molecular weights, with a mean value of 3.12 +/- 0.21 X 10(6) (range: 2.92 X 10(6) to 3.45 X 10(6)), and an average partial specific volume of 0.969 +/- 0.003 ml/g; both were assessed by flotation equilibrium analysis in the analytical ultracentrifuge. In the individual animals, however, the physical properties of LDL were invariant with time. The administration of either an "average" American diet or a coconut oil-cholesterol diet was accompanied by hypercholesterolemia associated with changes in LDL which were characterized by increases in molecular weight to 3.52 +/- 0.21 X 10(6) (average of nine monkeys) and in partial specific volume to 0.973 +/- 0.002 ml/g. These changes were particularly evident when the molecular weight of LDL from monkeys in the normolipidemic state was compared with that obtained from the same monkeys during the hyperlipidemic state. Chemical analyses revealed that the particles from the hyperlipidemic animals had a relatively higher cholesteryl ester content, a slight increase in phospholipids, and a marked decrease to nearly complete absence of triglycerides. The other lipoprotein components, protein, carbohydrate, free cholesterol, and fatty acids, did not vary significantly from those of control LDL. It is concluded that the administration of atherogenic diets causes structural changes in LDL which appear to be accounted for, at least in part, by changes in the composition of the lipid moiety. The changes in physical and chemical properties noted in the LDL of rhesus monkeys with experimentally induced hypercholesterolemia contrast with the apparent structurally normal LDL from rhesus monkeys with spontaneous hypercholesterolemia reported previously.     

Plasma lipoprotein changes attending the intravenous administration of Triton WR-1339 in normolipidemic dogs: preferential effect on high density lipoproteins

The nonionic detergent Triton WR-1339 was injected intravenously into normolipidemic dogs in a single dose of 150 mg/kg body weight followed by three other injections (75 mg/kg) on days 2, 6, and 12. The Triton produced a significant elevation of the plasma cholesterol of these animals, but not of their triglyceride levels, and profound changes of their plasma lipoproteins, particularly of the high density lipoprotein class. These changes were dependent on the concentration of Triton attained in plasma; when the levels were above 1.5 mg/ml, density gradient ultracentrifugation, electrophoretic, and chemical analyses indicated that an interaction between Triton and HDL had occurred. This interaction was attended by a gradual loss of the surface components of HDL, namely apoA-I, phospholipids, and unesterified cholesterol, and by the appearance of two cholesteryl ester-rich lipoproteins of d 1.019-1.024 g/ml and d 1.038-1.058 g/ml containing apoA-I and proteins with electrophoretic mobilities of apoB, apoE, and apoA-IV. At the time that these changes had occurred, the activities of the enzymes lecithin: cholesterol acyltransferase and post-heparin lipase were unaffected. When 125I-labeled apoA-I was injected intravenously into animals receiving Triton, the residence time of the radiolabeled protein in plasma increased from a control value of 3.1 days to 7.2 days. However, the apparent half-times of the radiolabeled apoA-I varied among the lipoprotein fractions it was associated with: d 1.119-1.159 g/ml, 5.28 days; d 1.019-1.024 g/ml, 7.55 days, and d 1.038-1.058 g/ml, 5.39 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in ITGB3 has sex-specific associations with plasma lipoprotein(a) and whole blood serotonin levels in a population-based sample

A recent genome-scan identified the Leu33Pro polymorphism in the 3 integrin (ITGB3) gene as a quantitative trait locus for whole blood serotonin level in a large Hutterite pedigree. Because both the Leu33Pro polymorphism and the serotonin system have been implicated in cardiovascular disease (CVD) risk and treatment response, we studied additional variation in ITGB3 and its relationship to intermediate phenotypes associated with CVD in the same population. We examined associations between 15 single nucleotide polymorphisms (SNPs) across ITGB3 and five CVD-related traits in the Hutterites: plasma levels of high density lipoprotein-cholesterol (HDL-c), triglycerides (TG), low density lipoprotein-cholesterol (LDL-c), and lipoprotein(a) [Lp(a)] and blood pressure or hypertension. Seven of these SNPs in ITGB3 were associated with whole blood serotonin. Among the intermediate CVD-related phenotypes, only Lp(a) was associated with multiple ITGB3 SNPs, five of which were also associated with serotonin. A sex-stratified analysis revealed that the association between ITGB3 and Lp(a) is present only in females, whereas the association between ITGB3 and serotonin is concentrated in males. Our results suggest that variation in ITGB3 in addition to Leu33Pro could contribute to susceptibility to CVD and serotonin in a sex-specific manner.