Phenotype-Driven Plasma Biobanking Strategies and Methods.

Biobank development and integration with clinical data from electronic medical record (EMR) databases have enabled recent strides in genomic research and personalized medicine. BioVU, Vanderbilt's DNA biorepository linked to de-identified clinical EMRs, has proven fruitful in its capacity to extensively appeal to numerous areas of biomedical and clinical research, supporting the discovery of genotype-phenotype interactions. Expanding on experiences in BioVU creation and development, we have recently embarked on a parallel effort to collect plasma in addition to DNA from blood specimens leftover after routine clinical testing at Vanderbilt. This initiative offers expanded utility of BioVU by combining proteomic and metabolomic approaches with genomics and/or clinical outcomes, widening the breadth for potential research and subsequent future impact on clinical care. Here, we describe the considerations and components involved in implementing a plasma biobank program from a feasibility assessment through pilot sample collection.

Comparison of protein immunoprecipitation-multiple reaction monitoring with ELISA for assay of biomarker candidates in plasma.

Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978-0.995) between 10 and 640 ng/mL for the target proteins spiked into a "mock plasma" matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67-0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.

SLC1A5 mediates glutamine transport required for lung cancer cell growth and survival.

PURPOSE: We have previously identified solute-linked carrier family A1 member 5 (SLC1A5) as an overexpressed protein in a shotgun proteomic analysis of stage I non-small cell lung cancer (NSCLC) when compared with matched controls. We hypothesized that overexpression of SLC1A5 occurs to meet the metabolic demand for lung cancer cell growth and survival. EXPERIMENTAL DESIGN: To test our hypothesis, we first analyzed the protein expression of SLC1A5 in archival lung cancer tissues by immunohistochemistry and immunoblotting (N = 98) and in cell lines (N = 36). To examine SLC1A5 involvement in amino acid transportation, we conducted kinetic analysis of l-glutamine (Gln) uptake in lung cancer cell lines in the presence and absence of a pharmacologic inhibitor of SLC1A5, gamma-l-Glutamyl-p-Nitroanilide (GPNA). Finally, we examined the effect of Gln deprivation and uptake inhibition on cell growth, cell-cycle progression, and growth signaling pathways of five lung cancer cell lines. RESULTS: Our results show that (i) SLC1A5 protein is expressed in 95% of squamous cell carcinomas (SCC), 74% of adenocarcinomas (ADC), and 50% of neuroendocrine tumors; (ii) SLC1A5 is located at the cytoplasmic membrane and is significantly associated with SCC histology and male gender; (iii) 68% of Gln is transported in a Na(+)-dependent manner, 50% of which is attributed to SLC1A5 activity; and (iv) pharmacologic and genetic targeting of SLC1A5 decreased cell growth and viability in lung cancer cells, an effect mediated in part by mTOR signaling. CONCLUSIONS: These results suggest that SLC1A5 plays a key role in Gln transport controlling lung cancer cells' metabolism, growth, and survival.

Development of a novel sandwich ELISA for measuring cell lysate ABCA1 protein levels.

ATP-binding cassette transporter A-1 (ABCA1) mediates the transfer of cellular cholesterol to lipid-poor apolipoproteins. Liver X receptors (LXRs) are regulators of cholesterol homeostasis that increase transcription of ABCA1. Synthetic LXR agonists developed to date have been shown to induce ABCA1 mRNA expression and increase reverse cholesterol transport. Unfortunately, there have been few options for quantitatively measuring ABCA1 protein levels, including a previously described competitive ELISA standardized to an ABCA1 peptide with a sensitivity of 80 ng/ml. To address this unmet need, we developed a novel sandwich ELISA standardized to full-length human recombinant ABCA1 protein with sensitivity of approximately 0.5 ng/ml. To determine if the sandwich ELISA had adequate sensitivity to detect LXR-induced increases in ABCA1, we utilized it to measure ABCA1 levels in untreated and LXR agonist-treated human (THP-1) macrophage cells and human peripheral blood mononuclear cells (PBMC). Data obtained from the ELISA demonstrated an approximately eightfold increase in ABCA1 levels in both macrophages as well as PBMC in response to LXR agonist treatment, and results were highly correlated with those obtained by immunoprecipitation and western blotting. Together, these results suggest that the sandwich ELISA may be a sensitive and effective method for quantitating ABCA1 protein levels.

Fenofibrate treatment increases human serum proprotein convertase subtilisin kexin type 9 levels.

Over the past several years, proprotein convertase subtilisin kexin type 9 (PCSK9) has gained significant attention as a key regulator of serum LDL-cholesterol (LDL-C) levels. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDL-C and cardiovascular risk. Our laboratory was the first to demonstrate that atorvastatin increases PCSK9 serum levels, an observation that has since been confirmed by at least two other groups. In light of these observations, we studied the effect of another common lipid-lowering medication, fenofibrate, on circulating PCSK9 protein levels in patients treated with fenofibrate or placebo for 12 weeks. We observed that fenofibrate (200 mg per day) significantly increased circulating PCSK9 levels by 25% compared with baseline. Placebo treatment, in comparison, had no effect on PCSK9 levels. Interestingly, fenofibrate-induced increases in serum PCSK9 levels were highly correlated with fenofibrate-induced changes in HDL-C and triglyceride levels, as well as with fenofibrate-induced changes in LDL-C levels. These results suggest an explanation for why fibrates do not achieve as much LDL-C lowering as might otherwise be expected and indicate that the addition of a PCSK9 inhibitor to fibrate therapy may result in additional beneficial LDL-C lowering.

The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells.

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B(4) (LTB(4)) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB(4), a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB(4) was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025-25ng/mL for LTB(4) and 0.1-25ng/mL for 5-HETE. The EIA method has a validated range covering 0.025-4ng/mL. When both assays were applied to analyze LTB(4) from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r=0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB(4) and 5-HETE measured by LC/MS/MS after ionophore stimulation (r=0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.

Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 microg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30min and the degradation of hepatic LDLR reached the maximum 2h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5min of protein injection and internalized PCSK9 was only barely detectable within 1h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.

An apolipoprotein A-I mimetic dose-dependently increases the formation of prebeta1 HDL in human plasma.

Prebeta1 HDL is the initial plasma acceptor of cell-derived cholesterol in reverse cholesterol transport. Recently, small amphipathic peptides composed of D-amino acids have been shown to mimic apolipoprotein A-I (apoA-I) as a precursor for HDL formation. ApoA-I mimetic peptides have been proposed to stimulate the formation of prebeta1 HDL and increase reverse cholesterol transport in apoE-null mice. The existence of a monoclonal antibody (MAb 55201) and a corresponding ELISA method that is selective for the detection of the prebeta(1) subclass of HDL provides a means of establishing a correlation between apoA-I mimetic dose and prebeta1 HDL formation in human plasma. Using this prebeta1 HDL ELISA, we demonstrate marked apoA-I mimetic dose-dependent prebeta1 HDL formation in human plasma. These results correlated with increases in band density of the plasma prebeta1 HDL, when observed by Western blotting, as a function of increased apoA-I mimetic concentration. Increased prebeta1 HDL formation was observed after as little as 1 min and was maximal within 1 h. Together, these data suggest that a high-throughput prebeta1 HDL ELISA provides a way to quantitatively measure a key component of the reverse cholesterol transport pathway in human plasma, thus providing a possible method for the identification of apoA-I mimetic molecules.

Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has gained attention as a key regulator of serum low density lipoprotein cholesterol (LDL-C) levels. This novel protease causes the degradation of hepatic low density lipoprotein receptors. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDL-C levels and cardiovascular risk. Previous studies have demonstrated that statins upregulate PCSK9 mRNA expression in cultured cells and animal models. In light of these observations, we studied the effect of atorvastatin on circulating PCSK9 protein levels in humans using a sandwich ELISA to quantitate serum PCSK9 levels in patients treated with atorvastatin or placebo for 16 weeks. We observed that atorvastatin (40 mg/day) significantly increased circulating PCSK9 levels by 34% compared with baseline and placebo and decreased LDL-C levels by 42%. These results suggest that the addition of a PCSK9 inhibitor to statin therapy may result in even further LDL-C decreases.

Analytical validation and biological evaluation of a high molecular-weight adiponectin ELISA.

BACKGROUND: Of the 3 circulating multimeric forms of adiponectin, the high-molecular-weight (HMW) form, as measured by size-exclusion and/or immunoblotting techniques, is a better index of insulin sensitivity for monitoring health and disease than is total adiponectin. We aimed to develop a simple ELISA to measure HMW adiponectin. METHODS: We pretreated serum or plasma samples with digestion solution containing proteinase K (Millipore, ESDS). HMW (Millipore, EZHMWA-64K) and total adiponectin (Millipore, EZHADP-61K) concentrations were measured in treated and untreated samples, respectively, from 108 individuals and from 20 morbidly obese patients before and at 1, 3, 6, and 12 months after gastric-bypass surgery. RESULTS: The ELISA has a dynamic range of 3-200 microg/L and a detection limit of 0.8 microg/L. Intraassay and interassay CVs were <4% and <10%, respectively. Sample-dilution curves paralleled the calibration curves. Fast protein liquid chromatography profiles of the proteinase K-treated samples revealed predominantly HMW adiponectin. Values for HMW adiponectin produced with this method are comparable with those obtained with Western blot analysis (y = 0.77x - 0.15; r = 0.96; n = 56). Body mass index (BMI)- and sex-related changes were more pronounced for HMW adiponectin and percentage of HMW adiponectin than for total adiponectin. HMW and total adiponectin increased after bypass surgery, but changes in HMW adiponectin were more pronounced and preceded changes in total adiponectin. CONCLUSION: This simple, rapid ELISA for HMW adiponectin recognizes the HMW isoform, produces results closely correlated with those obtained with Western blotting, and appears to better distinguish BMI-, sex-, and weight loss-associated differences than assays for total adiponectin.

Serum proprotein convertase subtilisin kexin type 9 is correlated directly with serum LDL cholesterol.

BACKGROUND: Proprotein convertase subtilisin kexin type 9 (PCSK9) is gaining attention as a key regulator of serum LDL-cholesterol (LDLC). This novel serine protease causes the degradation of hepatic LDL receptors by an unknown mechanism. In humans, gain-of-function mutations in the PCSK9 gene cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDLC and decreased cardiovascular risk. Relatively little is known about PCSK9 in human serum. METHODS: We used recombinant human PCSK9 protein and 2 different anti-PCSK9 monoclonal antibodies to build a sandwich ELISA. We measured PCSK9 and lipids in 55 human serum samples and correlated the results. We used the anti-PCSK9 antibodies to assay lipoprotein particle fractions separated by sequential flotation ultracentrifugation. RESULTS: Serum concentrations of PCSK9 ranged from 11 to 115 microg/L and were directly correlated with serum concentrations of LDLC (r = 0.45, P = 0.001) and total cholesterol (r = 0.50, P = 0.0003), but not with triglycerides (r = 0.15, P = 0.28) or HDL cholesterol concentrations (r = 0.13, P = 0.36). PCSK9 was not detectable in any lipoprotein particle fraction, including LDL. CONCLUSIONS: PCSK9 is present in human serum, likely not associated with specific lipoprotein particles. The circulating concentrations of human PCSK9 are directly correlated with LDL and total cholesterol concentrations.

Relationship of apolipoprotein A5 and apolipoprotein C3 levels to serum triglycerides in patients with type 2 diabetes.

BACKGROUND: The role of apolipoprotein A5 (ApoA5) in modulating triglyceride levels in humans is incompletely understood. Some researchers have reported modest positive correlations of ApoA5 with triglycerides while others have reported negative correlations. A recent report suggested that ApoA5 gene expression may be influenced by insulin. In type 2 diabetes, some groups have reported higher levels of ApoA5 compared to normals while others have reported lower levels. METHODS: To better understand the relationships between ApoA5, apolipoprotein C3 (ApoC3), and triglycerides in type 2 diabetes, ApoA5 levels were measured and correlated with triglyceride, insulin, and HbA1c levels. ApoC3 levels were measured and correlated with triglycerides. RESULTS: In patients with type 2 diabetes, ApoA5 levels were elevated compared to normals, with several patients having markedly increased levels confirmed by Western blotting. ApoA5 levels were positively correlated with triglycerides (r=0.60) but were not correlated with either HbA1c or serum insulin levels. ApoC3 levels were highly positively correlated with triglycerides (r=0.88). CONCLUSIONS: These data indicate that in patients with type 2 diabetes ApoA5 levels are positively correlated with triglycerides but are not correlated with HbA1c or insulin levels. ApoC3 levels are strongly positively correlated with triglycerides in these patients.

Alloxan is an inhibitor of O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase.

We have previously shown that streptozotocin (STZ) inhibits O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from proteins. In light of this observation, we explored the possibility that the diabetogenic toxin alloxan, an O-GlcNAc transferase (OGT) inhibitor, might also inhibit O-GlcNAcase. Alloxan inhibited islet O-GlcNAcase with a dose-response much like that of STZ. Similar to STZ, islet O-GlcNAcase was more susceptible to alloxan inhibition than was brain O-GlcNAcase. Alloxan directly inhibited recombinant O-GlcNAcase activity with a dose-response very similar to that of STZ. Subsequent LC/MS/MS analysis revealed that alloxan modified the tryptic digest pattern of the enzyme. One tryptic peptide LGCFEIAK(894-901) was modified by alloxan. Two other tryptic peptides, LDQVSQFGCR(158-167) and SFALLFDDIDHNMCAADK(168-185), both N-terminal active site peptides, were absent after alloxan treatment. Together, these data demonstrate that alloxan is an inhibitor of O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase, with inhibition corresponding to an altered tryptic digest pattern of N-terminal active site peptides.

The diabetogenic antibiotic streptozotocin modifies the tryptic digest pattern for peptides of the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase that contain amino acid residues essential for enzymatic activity.

Streptozotocin (STZ) inhibits O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from proteins. The active site of the enzyme was recently proposed to include aspartates 174, 175, and 177, with STZ inhibition via a transition state analog. We explored the effect of STZ on the tryptic peptide digest pattern of O-GlcNAcase. LC/MS/MS analysis demonstrated that STZ modified two areas of the enzyme. One peptide, LGCFEIAK (894-901), in a C-terminal region previously proposed to possess O-GlcNAcase activity, was methylated by STZ. Another peptide, EYEIEFIYIASPGLDITFSNPK (128-149), was detected only after treatment with STZ and was in an N-terminal region, overlapping a glutamate-rich area containing an adjacent phenylalanine residue. No covalent modification of this peptide could be demonstrated. Detection of this peptide after treatment with STZ was accompanied by the simultaneous inability to detect the nearby peptide KLDQVSQFGCR (157-167), which contains a cysteine residue recently shown to be essential for enzymatic activity. To determine which of the first two peptides might also be important for O-GlcNAcase activity, site-specific mutagenesis was performed. Mutation of the N-terminal phenylalanine and serine residues resulted in almost complete inhibition of activity. In contrast, mutation of conserved C-terminal glycine and cysteine residues caused little inhibition of enzymatic activity. Together, these data extend the region of the active site N-terminally and give independent evidence to support the idea that STZ inhibits O-GlcNAcase through formation of a transition state analog that resides in the active site of the enzyme and in doing so alters its conformation and ensuing tryptic digest pattern.

Definitive N-terminal protein sequence and further characterization of the novel apolipoprotein A5 in human serum.

BACKGROUND: Apolipoprotein A5 (ApoA5) originally gained attention as a regulator of serum triglyceride concentrations through transgenic mouse studies. Our group recently developed the first assay to quantify serum ApoA5 protein concentrations and demonstrated that they are increased by administration of a potent peroxisome proliferator-activated receptor-alpha agonist. METHODS: To better characterize the circulating ApoA5, the protein was purified from human serum, and a definitive N-terminal protein sequence was obtained. In light of previous observations that ApoA5 was present in VLDL and not LDL, plasma infranatant and intermediate-density lipoprotein (IDL) were analyzed for ApoA5. Because the mature protein contains a single unpaired cysteine, ApoA5 in human serum was immunoprecipitated, and its migration pattern was examined via Western blotting under reducing and nonreducing conditions to determine whether the protein circulates as a disulfide-linked homodimer or heterodimer. RESULTS: Definitive N-terminal protein sequences obtained from ApoA5 purified from human serum indicated that cleavage of the signal peptide occurs in vivo at the predicted site. We found ApoA5 in VLDL, HDL, and chylomicrons but not in LDL, IDL, or plasma infranatant. Under both reducing and nonreducing conditions, ApoA5 migrated mainly as a single band with a relative molecular mass (Mr) of approximately 39,000, indicating that the protein exists in serum as a monomer and not as a disulfide-linked homodimer or heterodimer. CONCLUSIONS: Our data help characterize ApoA5 by defining its lipoprotein particle distribution, by determining its N-terminal protein sequence, and by demonstrating that the mature protein circulates mainly as a monomer and not as a disulfide-linked homodimer or heterodimer.

Administration of a PPARalpha agonist increases serum apolipoprotein A-V levels and the apolipoprotein A-V/apolipoprotein C-III ratio.

Apolipoprotein A-V (apoA-V) first gained attention as a regulator of triglycerides through transgenic mouse studies. Furthermore, peroxisome proliferator-activated receptor alpha (PPARalpha) agonists such as fenofibrate increase apoA-V mRNA expression. Our group recently developed the first assay to quantitate serum apoA-V levels. Therefore, we sought to determine whether administration of a PPARalpha agonist would increase circulating apoA-V. Cynomolgus monkeys were dosed for 14 days with 0.3 mg/kg/day LY570977 L-lysine, a potent and selective PPARalpha agonist. Blood samples were drawn throughout the treatment period and after a 2 week washout. Administration of the PPARalpha agonist caused a 50% decrease in triglycerides that reversed at washout. Serum apoA-V concentrations increased 2-fold, correlated inversely with triglycerides, and were reversible at washout. The apoA-V/apoC-III ratio increased >2-fold, with this increase also reversible at washout. These data demonstrate for the first time that a PPARalpha agonist increases circulating apoA-V protein levels and the apoA-V/apoC-III ratio.

The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.

BACKGROUND: The recently discovered apolipoprotein A5 (ApoA5) is fast gaining attention as a key regulator of serum triglyceride concentrations. An ApoA5 mouse knock-out model produced an approximately fourfold increase in serum triglycerides, whereas a knock-in model with human ApoA5 produced 50-70% lower concentrations of mouse serum triglycerides. In addition, peroxisome proliferator-activated receptor-alpha agonists, which are used clinically to lower serum triglyceride concentrations, cause increased ApoA5 mRNA expression. Despite these compelling molecular biology data, relatively little is known about ApoA5 protein in human serum. METHODS: To better understand circulating concentrations and lipoprotein particle distribution of ApoA5, we expressed the recombinant human ApoA5 protein and raised antibodies against both the NH(2) and COOH termini. RESULTS: Using the above reagents, we demonstrate for the first time that ApoA5 is present in human serum, although at much lower concentrations than other apolipoproteins such as ApoA1. Using a dual-antibody sandwich ELISA that we developed, we observed ApoA5 concentrations in human serum ranging from 24 to 406 microg/L compared with approximately 1 g/L for ApoA1. We also examined the lipoprotein particle distribution of ApoA5 and found that ApoA5 was detectable in VLDL, HDL, and chylomicrons, but not LDL. CONCLUSIONS: These data demonstrate for the first time that ApoA5 is a secreted protein present in human serum and is associated with specific lipoprotein particles. In addition, our data indicate that the circulating concentration of human ApoA5 is very low compared with other apolipoproteins.