Assessing the Practical Differences in LDL-C Estimates Calculated by Friedewald, Martin/Hopkins, or NIH Equation 2: An Observation Cross-Sectional Study.

Objective: Low-density lipoprotein-cholesterol (LDL-C) remains a clinically important cholesterol target in primary prevention of atherosclerotic cardiovascular disease. The present study aimed to assess the practical differences among three equations utilized for the estimation of LDL-C: the Friedewald, the Martin/Hopkins, and the NIH equation 2. Methods: Blood lipid measurements from 4,556 noninstitutionalized participants, aged 12 to 80, were obtained from the 2017-2020 National Health and Nutrition Examination Survey study. We 1) assessed the differences between three calculated LDL-C estimates, 2) examined the correlations between LDL-C estimates using correlation coefficients and regression, and 3) investigated the degree of agreement in classifying individuals into the LDL-C category using weighted Kappa and percentage of agreement. Results: The differences in LDL-C estimates between equations varied by sex and triglyceride levels (p<0.001). Overall, the mean of absolute differences between Friedewald and Martin/Hopkins was 3.17 mg/dL (median=2.0, 95% confidence interval [CI] [3.07-3.27]). The mean of absolute differences between Friedewald and NIH Equation 2 was 2.08 mg/dL (median=2.0, 95% CI [2.03-2.14]). Friedewald correlated highly with Martin/Hopkins (r=0.991, rho=0.989) and NIH Equation 2 (r=0.998, rho=0.997). Cohen's weighted Kappa=0.92 between Friedewald and Martin/Hopkins, and 0.95 between Friedewald and NIH equation 2. The percentage of agreement in classifying individuals into the same LDL-C category was 93.0% between Friedewald and Martin/Hopkins, and 95.4% between Friedewald and NIH equation 2. Conclusion: Understanding the practical differences in LDL-C calculations can be helpful in facilitating decision-making during a paradigm shift.

The significance of arginase I administration on the survival of mice bearing NS-1 myeloma cells.

BACKGROUND: Arginase I blood levels elevate in cancerous patients and correlate with cancer stages and poor prognosis. Since arginase is capable of enhancing cell growth, it is unclear whether its ominous effect on cancer progression is through the inhibition of immunity or through direct enhancement of cancer cell growth. We tried to clarify this question. METHODS: NS-1 mouse myeloma cells were inoculated intraperitoneally (i.p.) into mice. Purified mouse arginase I was injected daily either intravenously (i.v.) or i.p. for 6 d. A tumor-only control group received i.p. tumor cells without arginase. The survival rates of all mice were recorded. RESULTS: Survival rates were significantly lower in the i.v. group than in the i.p. group (P=0.017) or in the tumor-only control group (P=0.034). As spleen is readily exposed to i.v. arginase, its natural killer cells were studied and were found to have been significantly suppressed by arginase in vitro (P<0.005). CONCLUSION: Our results indicate that the direct inhibition of the immune system by i.v. arginase is more significant in shortening the survival of tumor-bearing mice than localized (i.p.) arginase promotion of tumor cell growth. Thus, an elevation of arginase in a patient's blood is very harmful to the host immune system, e.g. splenic natural killer cells.

Fluorescence activated cell sorting (FACS) using RNAlater to minimize RNA degradation and perturbation of mRNA expression from cells involved in initial host microbe interactions.

Host microbe interactions frequently involve specific cellular tropism. Accurate characterization of cells involved in these initial interactions is complicated by the response to the microbe. We describe a method utilizing RNAlater for Fluorescence Activated Cell Sorting of these critical cells that minimizes the downstream perturbation in the gene expression profile.

Identification of chondrocyte proliferation following laser irradiation, thermal injury, and mechanical trauma.

BACKGROUND AND OBJECTIVE: Cartilage has a limited regenerative capacity, and there are a lack of reliable techniques and methods to stimulate growth of new tissue to treat degenerative diseases and trauma. This study focused on identifying chondrocyte cell proliferation in ex vivo cartilage tissue following heating Nd:YAG laser using whole-mount analysis and flow cytometry, and compared findings with results produced by contact, and water bath heating methods, mechanical injury, and the addition of transforming growth factor-beta (TGF-beta). STUDY DESIGN/MATERIALS AND METHODS: Ex vivo rabbit nasal septal cartilages were either irradiated with an Nd:YAG laser (lambda = 1.32 microm, 2-16 seconds, 6 W/cm(2)), heated by immersion in a warm saline bath, heated by direct contact with a metal rod, or mechanically damaged by scoring with a scalpel or crushing. After treatment, specimens were incubated for 7 or 14 days in growth media containing 10 microM bromodeoxyuridine (BrdU). Additional specimens were cultured with both BrdU and TGF-beta. Both whole-mount BrdU-double-antibody detection techniques and flow cytometry were used to determine the presence of DNA replication as a marker of proliferation. RESULT: An annular region of regenerating chondrocytes was identified surrounding the laser irradiation zone in whole-mount tissue specimens, and the diameter of this region increased with irradiation time. Using whole-mount analysis, no evidence of chondrocyte DNA replication was observed in tissues heated using non-laser methods, grown in TGF-beta, or mechanically traumatized. In contrast, flow cytometry identified the presence of BrdU-positive cells in the S-phase of the cell cycle (synthesis of DNA) for all protocols, indicating chondrocyte proliferation. The percentage of cells that are in S-phase increased with irradiation time. CONCLUSION: These data provide evidence that laser irradiation, along with other thermal and mechanical treatments, causes a proliferative response in chondrocytes, and this is observed ex vivo in the absence of cellular and humoral repair mechanisms. The advantage of using optical methods to generate heat in cartilage is that microspot injuries could be created in tissue and scanned across surfaces in clinical applications.

Lead exposure raises superoxide and hydrogen peroxide in human endothelial and vascular smooth muscle cells.

BACKGROUND: Chronic lead exposure causes hypertension and cardiovascular disease, which are associated with, and, in part, due to oxidative stress. While occurrence of oxidative stress in lead-exposed animals and cultured endothelial cells has been well-established, direct and specific evidence on the type of the reactive oxygen species (ROS) produced by lead-exposed vascular cells is lacking and was investigated. METHODS: Human coronary endothelial (EC) and vascular smooth muscle cells (VSMC) were incubated in appropriate culture media in the presence of either 1 ppm or 10 ppm lead acetate or sodium acetate (control) for 1 to 30 minutes or 60 hours. Productions of superoxide and hydrogen peroxide in the cell populations were determined by flow cytometry using hydroethidine and dihydrorhodamine, respectively. Data from a minimum of 10,000 cells were collected and analyzed using Cell Quest software. In addition, Cu Zn superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), and NAD(P)H oxidase (gp91phox) were measured. RESULTS: Short-term lead exposure resulted in a significant rise in both superoxide and hydrogen peroxide production by both EC and VSMC. After long-term exposure, detectable superoxide levels fell to near normal level, while hydrogen peroxide production remained high. This was associated with up-regulations of gp91phox, elevation of superoxide dismutase, reduction of VSMC catalase, and no change in GPX levels. Together, these events can account for the observed decline in superoxide and the rise in hydrogen peroxide following long-term lead exposure. CONCLUSION: Lead exposure promotes generation of superoxide and hydrogen peroxide in human EC and VSMC. This phenomenon can potentially contribute to the pathogenesis of the lead-associated hypertension and cardiovascular disease, and points to the potential benefit of lowering lead burden in the exposed populations.

A multiplex microsphere bead assay for comparative RNA expression analysis using flow cytometry.

Comparative gene-expression profiling is an important tool in understanding molecular signatures of complex diseases as well as the responses of cells and tissues to external factors. With increasing microarray data, disease-specific molecular patterns are emerging but the acquisition of these data is expensive, difficult to customize and not well standardized. Once genome-wide scans identify differentially expressed genes in a given disease, cheaper, more easily customized methods will be needed for evaluating the expression of these genes in large population samples. Here we describe a novel multiplex microsphere bead assay (MBA) to compare gene expression levels. To test this assay we evaluated the expression levels of four transcripts (BRCA1, MGB1, DLG1 and ACT1) in normal and cancerous mammary tissue. The results were consistent with those generated by quantitative real-time PCR.

Deficiency of the Nrf1 and Nrf2 transcription factors results in early embryonic lethality and severe oxidative stress.

Nrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. We previously showed that loss of Nrf1 function in mice results late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, we generated mice that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, we show that expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells.