Potential predictive biomarkers of adalimumab response in patients with hidradenitis suppurativa.

BACKGROUND: Adalimumab provides significant efficacy for patients with hidradenitis suppurativa (HS), which was demonstrated by at least 50% of patients achieving a clinical response by week 12 that was maintained through to week 168 in the PIONEER trials. OBJECTIVES: To identify whether there are biomarkers that could predict adalimumab response, as well as markers that differentially respond to adalimumab in patients with HS. METHODS: Baseline and week-12 plasma samples from the PIONEER studies were used to assess the levels of circulating proteins by multiplex and enzyme-linked immunosorbent assays. RESULTS: Analyses revealed significantly higher high-sensitivity C-reactive protein (hs-CRP) and chemokine (C-C motif) ligand (CCL) 16 (HCC-4) levels in nonresponders at baseline and identified a multivariate response signature of calprotectin, fractalkine and HCC-4, reaching an 86% predictive accuracy rate for adalimumab response. Additionally, post-treatment reduction of plasma C-X-C motif chemokine ligand (CXCL)9, CXCL8 (interleukin-8) and CCL19 (macrophage inflammatory protein 3ß) were greater in adalimumab super-responders than in nonresponders (P = 0·026, P = 0·044 and P = 0·026, respectively). These cytokines are involved in the recruitment of innate and adaptive inflammatory cells, and/or stimulation of certain inflammatory responses, suggesting that these pathways could be disease drivers in adalimumab nonresponders. CONCLUSIONS: These initial results suggest HCC-4, calprotectin and fractalkine could be potential predictive biomarkers of adalimumab response in HS and identified possible tumour necrosis factor-independent disease pathways.

Deletion of murine Arv1 results in a lean phenotype with increased energy expenditure.

BACKGROUND: ACAT-related enzyme 2 required for viability 1 (ARV1) is a putative lipid transporter of the endoplasmic reticulum that is conserved across eukaryotic species. The ARV1 protein contains a conserved N-terminal cytosolic zinc ribbon motif known as the ARV1 homology domain, followed by multiple transmembrane regions anchoring it in the ER. Deletion of ARV1 in yeast results in defective sterol trafficking, aberrant lipid synthesis, ER stress, membrane disorganization and hypersensitivity to fatty acids (FAs). We sought to investigate the role of Arv1 in mammalian lipid metabolism. METHODS: Homologous recombination was used to disrupt the Arv1 gene in mice. Animals were examined for alterations in lipid and lipoprotein levels, body weight, body composition, glucose tolerance and energy expenditure. RESULTS: Global loss of Arv1 significantly decreased total cholesterol and high-density lipoprotein cholesterol levels in the plasma. Arv1 knockout mice exhibited a dramatic lean phenotype, with major reductions in white adipose tissue (WAT) mass and body weight on a chow diet. This loss of WAT is accompanied by improved glucose tolerance, higher adiponectin levels, increased energy expenditure and greater rates of whole-body FA oxidation. CONCLUSIONS: This work identifies Arv1 as an important player in mammalian lipid metabolism and whole-body energy homeostasis.

Predicting the sites of metastases from lung cancer using molecular biologic markers.

BACKGROUND: The use of molecular markers in staging non-small cell lung cancer (NSCLC) has been supported in retrospective prognostic models but has not been evaluated in predicting sites of metastases. METHODS: Pathologic specimens were collected from 202 patients after complete resection for stage I NSCLC, who were subsequently found to have no metastases at 5 years (n = 108), isolated brain metastases (n = 25), or other distant metastases (n = 69). A panel of eight molecular markers of metastatic potential was chosen for immunohistochemical analysis of the tumor: p53, erbB2, angiogenesis factor viii, EphA2, E-cadherin, urokinase plasminogen activator (UPA), UPA receptor, and plasminogen activator inhibitor. RESULTS: Patients with isolated brain relapse had significantly higher expression of p53 (p = 0.02) and UPA (p = 0.002). The quantitative expression of E-cadherin was used to predict the site of metastases using recursive partitioning: 0 of 92 patients with E-cadherin expression of 0, 1, or 2 developed isolated cerebral metastases; 0 of 33 patients with E-cadherin expression of 3 with UPA of 1 or 2 and ErbB2 of 0 developed brain metastases. Of the remaining patients at risk (UPA = 3), the risk of isolated cerebral metastases was 21 of 57 patients (37%). CONCLUSIONS: This study demonstrates that molecular markers may predict the site of relapse in early stage NSCLC. If validated in an ongoing prospective study, these results could be used to select patients with isolated brain metastases for adjuvant therapy, such as prophylactic cranial irradiation.

Justice at the millennium: a meta-analytic review of 25 years of organizational justice research.

The field of organizational justice continues to be marked by several important research questions, including the size of relationships among justice dimensions, the relative importance of different justice criteria, and the unique effects of justice dimensions on key outcomes. To address such questions, the authors conducted a meta-analytic review of 183 justice studies. The results suggest that although different justice dimensions are moderately to highly related, they contribute incremental variance explained in fairness perceptions. The results also illustrate the overall and unique relationships among distributive, procedural, interpersonal, and informational justice and several organizational outcomes (e.g., job satisfaction, organizational commitment, evaluation of authority, organizational citizenship behavior, withdrawal, performance). These findings are reviewed in terms of their implications for future research on organizational justice.

Chromophore-modified bis-naphthalimides: synthesis and antitumor activity of bis-dibenz[de,h]isoquinoline-1,3-diones.

The bis-dibenz[de,h]isoquinoline-1,3-diones are a new series of antitumor agents that consist of two chromophores bridged by an alkylamino linker. In the present study we have explored the effect produced by the presence of two dibenz[de,h]isoquinoline-1,3-dione moieties with different polyamine chains on cellular cytotoxicity. Bis-dibenz[de,h]isoquinoline-1,3-diones with the bridge (CH2)2-NH-(CH2)n-NH-(CH2)2, where n = 2-5, showed optimum cytotoxicity with IC50's around 10 nM. Compound 16, which has the (CH2)2-NH-(CH2)3-NH-(CH2)2 bridge, altered DNA mobility and topoisomerase I and II activity at approximately 5 microM. When tested in vivo, compound 16 increased the median survival time of mice implanted with M5076 with an optimum %T/C of 154% and produced cures in 50% of mice implanted with Lox melanoma.

Mechanistic aspects of chiral discrimination on modified cellulose.

Cellulose and cellulose derivatives are biopolymers which are often used as stationary phases for the separation of enantiomers. Describing the mechanism of such separations is a difficult task due to the complexity of these phases. In the present study, we attempt to elucidate the types of interactions occurring between a diol intermediate for a LTD(4) antagonist and a tris(4-methylbenzoate)-derivatized cellulose stationary phase. Thermodynamic studies indicate that, at low temperatures, the enantioselectivity is entropy driven. At higher temperatures, the separation is enthalpy driven. DSC and IR experiments reveal that the transitions between the enthalpic and the entropic regions of the van't Hoff plots are a result of a change in conformation of the stationary phase. Investigation of chromatographic kinetic parameters reveals that, at low temperature, the second eluted enantiomer undergoes sluggish inclusion interactions. Subtle changes in the structure of the analyte indicates that π-π interactions do not contribute to enantioselectivity. Finally, molecular modeling of (R)- and (S)-diol and the stationary phase suggests that hydrogen bonding is a primary factor in the separation, and the calculated energy values obtained from the molecular modeling correlate well with the chromatographic elution order.

Prognostic value of MIB-1 in advanced ovarian carcinoma as determined using automated immunohistochemistry and quantitative image analysis.

BACKGROUND AND OBJECTIVES: The monoclonal antibody MIB-1 is an immunohistochemical marker reacting most strongly with cells in late S phase, G2, and M portions of the cell cycle. This antibody, reactive in formalin-fixed, paraffin-embedded tissue, allows the quantitation of a proliferation index (PI) in both current clinical cases and archival material using a computerized image analyzer (CIA). METHODS: Since many laboratories make use of automated immunohistochemistry (AIH), this study was performed to explore the technical feasibility of using AIH (Ventana ES 320) in combination with CIA (CAS 200) to evaluate MIB-1 PI as a prognostic marker as assessed by overall survival in 50 archival (formalin-fixed, paraffin-embedded), advanced stage primary ovarian carcinomas. RESULTS: Exploratory methods confirmed that 15% was a cutpoint that could dichotomize these 50 patients into two prognostic groups based on overall survival. The median survival of patients whose carcinoma had a high MIB-1 expression (> or = 15%) was 16 months compared with 30 months in the patients whose tumors demonstrated low MIB-1 expression (< 15%, P = 0.01). After adjustment for age, MIB-1 retained its prognostic significance (P = 0.02). Patients 60 years and older had shorter survival than younger patients (P = 0.06), but these two groups did not differ with respect to PI (P = 0.76). Those patients with a negative second look laparotomy had a longer median survival of 70 months compared with 18.5 months in patients with a positive second look (P < 0.001); the median PIs were 17% and 27%, respectively (P = 0.36). There were no significant relationships between clinical stage, nuclear grade, DNA ploidy, p53, and either survival or PI. CONCLUSIONS: In this study, we concluded that the combination of AIH and CIA yielded a reliable quantitation of MIB-1 proliferative index and that this proliferative marker had prognostic significance in late stage ovarian carcinoma. Further studies in a larger group of patients are needed to confirm the relationship between proliferation index and other known clinicopathologic and genetic variables.

Bis-naphthalimides 3: synthesis and antitumor activity of N,N'-bis[2-(1,8-naphthalimido)-ethyl] alkanediamines.

The bis-naphthalimides are a new class of antitumor agent. Previously, we have described initial synthetic series which established that two naphthalimide chromophores joined by polyamine linkers can produce agents with antitumor activity. We now extend these studies to describe the synthesis of a N,N'-bis[1,8-naphthalimido)-ethyl]alkanediamine series and examine the alkyl linker and chromophore substitution requirements for optimal antitumor activity. As a result, a bis-naphthal-imidoethyl derivative with no chromophore substitution and a NH-(CH2)3-NH bridge was identified. This agent (LU 79553) had both potent cellular cytotoxicity (IC50 = 0.014 microM) and was curative against MX-1 tumors grown in athymic mice. It has now been selected for clinical development.

Estrogen and progesterone receptor status determined by the Ventana ES 320 automated immunohistochemical stainer and the CAS 200 image analyzer in 236 early-stage breast carcinomas: prognostic significance.

The quantitation of estrogen and progesterone receptors (ER and PgR) has become the standard of care in the evaluation of patients with primary breast carcinoma. It has been demonstrated that ER and PgR detected by immunohistochemical methods in formalin-fixed paraffin-embedded tissue can be quantified by computerized image analysis. In this study, ER and PgR levels were determined by using an automated immunochemistry stainer (Ventana ES 320) and an image analyzer (CAS 200) in a series of 236 patients with stage I/II carcinoma of the breast. The degree of correlation of the ER and PgR levels determined by the dextran-coated charcoal method (DCC) with image analysis quantitation was high (r=0.75). The agreement between both methods was 77% for ER and 73% for PgR. Hormone receptor levels were correlated with prognosis as determined by overall survival. An ER level of 30 fmol/mg as determined by image analysis was established to stratify the patient population most effectively into favorable and unfavorable prognostic groups (P=0.003). An ER level of 20 fmol/mg for prognostic stratification reached statistical significance (P=0.03). The DCC method was not able to stratify the patients into prognostic groups at the traditionally accepted cutpoint of 10 fmol/mg (P=0.52). We conclude that when used in combination, automated immunohistochemistry and quantitative image analysis offer a favorable alternative to the DCC method in assessment of ER and PgR status in human mammary carcinoma. In addition, quantitative immunocytochemistry techniques may prove superior to the DCC method in specimens in which there is limited tumor volume (including fine-needle aspirates), stroma-rich tumors, and early-stage lesions including intraductal carcinoma.

Effect of ageing and propranolol administration on myocardial beta-adrenoceptor receptor function in mature rats.

Ageing is accompanied by diminishing myocardial tissue beta-adrenoceptor responses. The relative contribution of maturation and senescence to reported age-related changes in cell-surface beta-adrenoceptor dysfunction has not been established, since previous investigation has incorporated young rats lacking full maturity. We have examined myocardial ventricle membrane beta-adrenoceptor function in mature young (6 month) and old (26 month) male Wistar rats and the effect of propranolol infusion for seven days on beta-adrenoceptor function in these groups. beta 1-adrenoceptors comprised 63-72% of total beta-adrenoceptor density in both groups. beta 1-adrenoceptor densities were similar in young and old rats (young, 20.4 +/- 2.3; old, 24.7 +/- 1.4 fmol/mg protein +/- S.E.). beta 2-adrenoceptor densities were higher in older rats (young, 8.2 +/- 0.5, n = 9; old, 13.6 +/- 1.8, n = 9 fmol/mg protein +/- S.E., P < 0.025). Subcutaneous infusion of propranolol for seven days with miniosmotic pumps was accompanied by an increase in beta 1- and beta 2-adrenoceptor densities in young rats only (beta 1-, 38%, P < 0.05; beta 2- 52%, P < 0.025). beta 1-adrenoceptor agonist affinity and adenylate cyclase response to isoprenaline, GTP, Gpp(NH)p, Mn2+ and forskolin were not affected by age or propranolol infusion in either age-group. These findings demonstrate that male Wistar rats do not exhibit changes in myocardial ventricle beta-adrenoceptor-G-protein coupling capacity or adenylate cyclade activation with ageing beyond maturity.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical evaluation of LU 79553: a novel bis-naphthalimide with potent antitumor activity.

LU 79553 is a novel bis-naphthalimide which is highly cytotoxic in vitro with EC50 (concentration required for 50% inhibition of growth) ranging from 2 x 10(-7) to 5 x 10(-10) M. A number of studies were conducted to examine its antitumor activity in human xenograft models. In addition, we wanted to explore the possible schedule dependency of LU 79553 cytotoxicity in these xenograft models. Complete regression of MX-1 (mammary carcinoma) xenografts was observed when LU 79553 was administered i.v. daily for 5 doses at 20 mg/kg (2 cycles starting on Days 6 and 20) or every 3 days for 2 doses at 55 mg/kg (2 cycles starting on Days 6 and 13) or every 7 days for 4 doses. Complete regression was also seen in the MX-1 model when tumors were staged at 1-2 g prior to the initiation of treatment. Regressions (complete or partial) were observed in the LX-1 (lung), CX-1 (colon), DLD (colon), and LOX (melanoma) xenograft models. A significant increase in the median survival time of OVCAR-3- (ovarian carcinoma) bearing mice was noted in LU 79553-treated animals (treated/control = 195%). The excellent activity of this compound in such a wide variety of tumor types suggests LU 79553 merits further investigation in clinical trials.

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins, osteocalcin, and matrix GLA protein in vitro.

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, gamma-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 micrograms/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes.

Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes.

To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoids promote changes in gene expression involved in cell-cell and cell-extracellular matrix signaling mechanisms that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue-like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.

Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue-like organization in primary osteoblast cultures.

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue-like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue-like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H-thymidine labelling and autoradiography, indicate that greater than 98% of the pre-confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down-regulated cell growth associated genes. Post-proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial-derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue-like organization.

The effect of donor age on human fibroblast beta-adrenergic receptor response and agonist-induced desensitization.

Aging has been associated with changes in beta-adrenergic receptor (beta-receptor) function in several tissues. The relative contribution of cellular aging and age-related changes in homeostatic regulation of receptor function is unknown. We have examined beta-receptor function in fibroblasts of young and old donors (young: mean age 31.2 +/- 0.8 years +/- S.E., n = 6; old: mean age 81.8 +/- 0.6 years +/- S.E., n = 6). Beta-receptor responses to isoproterenol (ISO), (1 microM) were similar in the two groups. The concentration of ISO required for 50% maximal beta-receptor-mediated cyclic AMP production (EC50) was similar in both groups. Fibroblast beta-receptor density was also similar in young and old groups. ISO-induced beta-receptor desensitization was both dose- and time-dependent. Submaximal desensitization by acute exposure (30 min) to ISO (1 mM) caused similar levels of beta-receptor desensitization in young (42.5 +/- 2.5%) and old (42.8 +/- 2.8%) groups and a similar increase in ISO EC50. These findings demonstrate that aging in vivo does not cause changes in fibroblast beta-receptor regulation that are retained in culture.

Hepatic beta-adrenoceptor adaptation during propranolol administration is impaired in aging rats.

Aging has been associated with changes in beta-adrenoceptor responses and adaptation to prolonged removal of catecholamine stimulation. We have examined the effect of chronic propranolol administration on rat hepatic membrane beta-adrenoceptor density, agonist affinity and response in young (6-7 months) and old (26-7 months) male Wistar rats. Propranolol administration via miniosmotic pumps for 7 days resulted in similar and sustained plasma propranolol levels (approximately 100 ng/ml) in old and young rats. Pretreatment beta-adrenoceptor responses to isoprenaline were significantly higher in old rats. Propranolol administration was associated with significant increases in beta-adrenoceptor response and density (Bmax) in young rats only. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) responses to prostaglandin E1 (PGE1), guanosine triphosphate (GTP), 5'-guanyl-imidodiphosphate (Gpp(NH)p), forskolin and Mn2+ were not significantly different between young and old rats and were not affected by propranolol administration. Neither aging or propranolol administration was associated with a change in beta-adrenoceptor agonist affinity. These findings demonstrate elevated hepatic beta-adrenoceptor response and impaired hepatic beta-adrenoceptor adaptation to beta-adrenoceptor blockade in aging rats.

The use of visual information by house flies, Musca domestica (Diptera: muscidae), foraging in resource patches.

House flies, Musca domestica, respond to visual contrasts on the substrate if a resource is associated with the contrasting patterns. Visible resource patch boundaries serve as a signal to flies that they are about to leave a rewarding patch. Searching flies respond to such visual information by walking along the resource patch boundary and turning back into the patch at its edge. This edge detection and response serve as a mechanism for flies with visual cues to stay in a rewarding patch and locate more resources within it. The intensity of their response correlates with the quality of the resource. In the absence of visual cues, patch shape affects foraging success; flies find more resources in circular than in linear resource distributions. The effects of visual cues, however, render patch shape unimportant. Various substrate contrasts are effective as resource information for flies: dark (e.g., green) figures on bright (e.g., white) backgrounds or bright figures on dark backgrounds. Responses to substrate contrasts measured in this study indicate that, over the short term, house flies can learn a visual cue associated with a food source.

Quantitative determination of high-density lipoprotein cholesterol by agarose gel electrophoresis.

We have developed a procedure for the determination of high-density lipoprotein cholesterol by agarose gel electrophoresis. Only 2 micro L of sample was applied to the gel. After electrophoresis at 90 V for 35 min, an enzymatic cholesterol reagent was applied. After a 30-min incubation, the high-density lipoprotein cholesterol was quantified by densitometry. Precision for this measurment approaches that reported for the heparin-manganese/Abell-Kendall method (Clin. Chem, 25: 596--609, 1979). We evaluated accuracy by comparing high-density lipoprotein cholesterol concentration measured by electrophoresis to that determined in the Framingham Heart Study procedure (J. Biol. Chem. 195: 357, 1952). The resulting correlation was excellent. By the paired Student's t-test, there was no significant difference between the two methods. The proposed method gives a linear standard curve when the concentration of total cholesterol is between 1.0 and 3.5 g/L. By accurate quantitation of high-density lipoprotein cholesterol, agarose gel electrophoresis can aid in assessment of coronary heart disease risk for a large segment of the population.