Infrapatellar fat pad abnormalities are associated with a higher inflammatory synovial fluid cytokine profile in young adults following ACL tear.

OBJECTIVE: To evaluate the degree of knee fat pad abnormalities after acute anterior cruciate ligament (ACL) tear via magnetic resonance fat pad scoring and to assess cross-sectionally its association with synovial fluid biomarkers and with early cartilage damage as quantified via T1ρ and T2 relaxation time measurements. DESIGN: 26 patients with acute ACL tears underwent 3T MR scanning of the injured knee prior to ACL reconstruction. The presence and degree of abnormalities of the infrapatellar (IPFP) and the suprapatellar (SPFP) fat pads were scored on MR images along with grading of effusion-synovitis and synovial proliferations. Knee cartilage composition was assessed by 3T MR T1ρ and T2 mapping in six knee compartments. We quantified concentrations of 20 biomarkers in synovial fluid aspirated at the time of ACL reconstruction. Spearman rank partial correlations with adjustments for age and gender were employed to evaluate correlations of MR, particularly cartilage composition and fat pad abnormalities, and biomarker data. RESULTS: The degree of IPFP abnormality correlated positively with the synovial levels of the inflammatory cytokine markers IFN-γ (ρpartial = 0.64, 95% CI (0.26-0.85)), IL-10 (ρpartial = 0.47, 95% CI (0.04-0.75)), IL-6 (ρpartial = 0.56, 95% CI (0.16-0.81)), IL-8 (ρpartial = 0.49, 95% CI (0.06-0.76)), TNF-α (ρpartial = 0.55, 95% CI (0.14-0.80)) and of the chondrodestructive markers MMP-1 and -3 (MMP-1: ρpartial = 0.57, 95% CI (0.17-0.81); MMP-3: ρpartial = 0.60, 95% CI (0.21-0.83)). IPFP abnormalities were significantly associated with higher T1ρ and T2 values in the trochlear cartilage (T1ρ: ρpartial = 0.55, 95% CI (0.15-0.80); T2: ρpartial = 0.58, 95% CI (0.18-0.81)) and with higher T2 values in the medial femoral, medial tibial as well as in patellar cartilage (0.45 ≤ ρpartial ≤ 0.59). Correlations between SPFP abnormalities and synovial markers were not significant except for IL-6 (ρpartial = 0.57, 95% CI (0.17-0.81)). CONCLUSIONS: This exploratory study suggests that acute ACL rupture can be associated with damage to knee tissues such as the inferior fat pad of the knee. Such fat pad injury could be partially responsible for the apparent post-injury pro-inflammatory response noted in ACL-injured individuals. However, future longitudinal studies are needed to link ACL-rupture associated fat pad injury with important patient outcomes such as the development of posttraumatic osteoarthritis.

Chondroitin sulphate inhibits NF-κB activity induced by interaction of pathogenic and damage associated molecules.

OBJECTIVE: To evaluate the anti-inflammatory mechanism of action of Chondroitin Sulphate (CS). DESIGN: THP-1 macrophages were cultured with a range of sizes and concentrations of HA fragments with TLR4 (LPS in a physiologically relevant concentration determined by analyses of sera of a community clinic ascertained knee osteoarthritis (OA) cohort) or TLR2 (heat killed listeria bacteria) agonists and varying concentrations of CS in a physiologically relevant range (10-200 µg/ml). We measured IL-1ß release, intracellular IL-1ß, proIL-1ß, caspase-1 and NF-κB activity and DNA binding activity of NF-κB transcription factors from nuclear and cytoplasmic extracts. RESULTS: Serum LPS was significantly associated with radiographic knee joint space narrowing (JSN) (P = 0.02) in the OA cohort (n = 40). The priming dose of LPS used for these experiments (10 ng/ml) was below the lowest serum concentration of the OA cohort (median 47.09, range 14.43-81.36 ng/ml). Priming doses of LPS and HA fragments alone did not elicit an inflammatory response. However, primed with LPS, HA fragments produced large dose-dependent increases in IL-1ß that were inhibitable by CS. CS did not inhibit caspase-1 activity but in physiologically achievable concentrations, attenuated NF-κB activity induced by either the TLR4 (LPS 1000 ng/ml) or TLR2 agonists alone or in combination with HA fragments. LPS induced and CS significantly reduced activity of canonical NF-κB transcription factors, p65, p50, c-Rel and RelB. CONCLUSIONS: Subinflammatory concentrations of pathogenic (LPS, listeria) and damage associated (HA) molecules interact to induce macrophage-related inflammation. CS works upstream of the inflammasome by inhibiting activation of NF-κB transcription factors.

Direct in vivo evidence of activated macrophages in human osteoarthritis.

OBJECTIVE: Through binding to folate receptor-ß (FR-ß), the new (99m)Tc-EC20 (Etarfolatide) imaging technique detects activated but not resting macrophages in vivo. The goal of this study was to investigate macrophage-related inflammation in osteoarthritis (OA). METHODS: Twenty-five individuals (50 knees) with symptomatic OA of at least one knee underwent SPECT-CT imaging of both knees and planar imaging of the whole body after injection of Etarfolatide. Scans and knee radiographs were scored blinded to clinical information including knee and other joint site pain severity. Measures of association controlled for age, gender, body mass index (BMI) and employed repeated measures to adjust for correlation between knees. DESIGN: Activated macrophages were present in the majority (76%) of knees. The quantity of knee-related macrophages was significantly associated with knee pain severity (R = 0.60, P < 0.0001) and radiographic knee OA severity including joint space narrowing (R = 0.68, P = 0.007), and osteophyte (R = 0.66, P = 0.001). Macrophages were also localized to joints commonly affected by OA including hand finger joints (12%), thumb bases (28%), shoulders (26%), great toes (18%) and ankles (12%). The presence of joint pain at fingers, wrists, ankles and great toes was significantly positively associated with presence of activated macrophages at these sites (P < 0.0001-0.04). CONCLUSIONS: This study provides the first direct in vivo evidence for macrophage involvement in OA in a substantial proportion of human knees. The association of quantity of activated macrophages with radiographic knee OA severity and joint symptoms suggests that drugs targeting macrophages and macrophage-associated inflammatory pathways may have the potential to be both symptom and structure modifying.

TSG-6 activity as a novel biomarker of progression in knee osteoarthritis.

OBJECTIVE: To establish whether there is an association between TSG-6 activity and osteoarthritis progression. DESIGN: TSG-6 activity was determined in 132 synovial fluids from patients with OA of the knee, using a novel quantitative TSG-6 activity assay. The association between TSG-6 activities at baseline and four distinct disease progression states, determined at 3-year follow-up, was analyzed using logistic regression. RESULTS: There was a statistically significant relationship between TSG-6 activity at baseline and all OA progression states over a 3-year period. Patient knees with TSG-6 activities in the top tenth percentile, compared to the median activity, had an odds ratio (OR) of at least 7.86 (confidence interval (CI) [3.2, 20.5]) for total knee arthroplasty (TKA) within 3 years, and of at least 5.20 (CI [1.8, 13.9]) after adjustment for confounding factors. Receiver operating characteristic (ROC) analysis for knee arthroplasty yielded a cut-off point of 13.3 TSG-6 activity units/ml with the following parameters: area under the curve 0.90 (CI [0.804, 0.996]), sensitivity 0.91 (CI [0.59, 0.99]), specificity 0.82 (CI [0.74, 0.88]) and a negative predictive value (NPV) of 0.99 (CI [0.934, 0.994]). CONCLUSION: The TSG-6 activity is a promising independent biomarker for OA progression. Given the high NPV, this assay may be particularly suitable for identifying patients at low risk of rapid disease progression and to assist in the timing of arthroplasty.

Effects of intraarticular IL1-Ra for acute anterior cruciate ligament knee injury: a randomized controlled pilot trial (NCT00332254).

OBJECTIVE: To evaluate the clinical effectiveness of intraarticular IL-1 receptor antagonist (IL-1Ra) for anterior cruciate ligament (ACL) tear. METHODS: Eleven patients with acute ACL tear confirmed by magnetic resonance imaging (MRI) were randomized to receive a single intraarticular injection of IL-1Ra (anakinra 150 mg, n = 6) or equal volume of saline placebo (1 ml, n = 5). The double-blinded treatment was administered a mean 2 weeks after injury. Synovial fluid (SF) (n = 9 patients) and sera (all patients) were available at baseline (prior to injection) and immediately prior to surgery (mean 35 days later) and analyzed for SF IL-1α, IL-1ß, IL-1Ra and serum hyaluronan (HA), an indicator of synovial inflammation. The primary outcome, standardized Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire, was obtained at 0 (baseline), 4, and 14 days after injection. RESULTS: Compared with placebo, the IL-1Ra group had substantially greater improvement in key outcomes over 14 days (KOOS pain P = 0.001; activities of daily living P = 0.0015; KOOS sports function P = 0.0026; KOOS quality of life (QOL) P = 0.0048; and total KOOS P < 0.0001). There were no adverse reactions in either group. SF IL-1α (P = 0.05) and serum HA (P = 0.03), but not IL-1ß, or IL-1Ra, decreased significantly in the IL-1Ra but not the placebo treated patients. Compared with placebo, IL-1α was borderline significantly different in the IL-1Ra treated group (P = 0.06). CONCLUSIONS: Administered within the first month following severe knee injury, IL-1Ra reduced knee pain and improved function over a 2-week interval. This promising proof of concept study provides a new paradigm for studies of acute joint injury and suggests that a larger follow-up study is warranted.

Selenomethionine inhibits IL-1ß inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) expression in primary human chondrocytes.

OBJECTIVE: Several lines of evidence show that selenium (Se) has potential protective effects in osteoarthritis (OA), however the exact mechanism is still unclear. As interleukin-1ß (IL-1ß) is one of the key proinflammatory cytokines contributing to the progression in OA, we investigated the effect of Se in neutralizing the inflammatory effects of IL-1ß on nitric oxide (NO) and prostaglandin E2 (PGE2) production, and the signaling pathways involved. METHODS: Isolated primary human chondrocytes were pretreated with selenomethionine (SeMet) (0.5 µM SeMet) for 24 h then co-treated without or with IL-1ß (10 pg/ml or 50 pg/ml) for another 24 h followed by RNA isolation. Gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) was determined by quantitative Real Time-Polymerase Chain Reaction. Culture media concentrations of NO and PGE2 were determined by nitrite (NO2⁻) assay and immunoassay respectively. For analysis of cell signaling pathways, chondrocytes were pretreated with SeMet then stimulated with IL-1ß for 0-45 min. The activity of IL-1ß signaling pathways was determined by Western blot screening of phosphorylation states of signal transduction proteins. RESULTS: SeMet inhibited chondrocyte gene expression of IL-1ß induced iNOS (31-54%, P=0.031) and COX2 (50-65%, P=0.031) with corresponding reductions in both NO (19-47%, P=0.031) and PGE2 (24-32%, P=0.031) production. Pretreatment with SeMet attenuated IL-1ß induced activation of p38 MAPK (39%, P=0.039) but not the extracellular signal-regulated kinase pathways (ERK) 1/2, c-Jun N-terminal kinases (JNK) or nuclear factor κB (NFκB). CONCLUSIONS: This study elucidates one potential protective mechanism of Se, namely through the alteration of cell signaling and downstream transcription of pro-inflammatory effects of IL-1ß.

Serum cartilage oligomeric matrix protein (COMP) level is a marker of disease activity in relapsing polychondritis.

OBJECTIVES: Relapsing polychondritis (RP) is a rare and severe disease which may lead to destruction of elastic cartilages. Until now, no reliable biomarker of disease activity in RP has been available. This study was designed to measure serum levels of cartilage biomarkers during both active and inactive phases of the disease. METHODS: Serum levels of cartilage oligomeric matrix protein (COMP), chondroitin sulfate 846 epitope (CS846) of proteoglycan aggrecan and collagen type II collagenase cleavage neoepitope (C2C) were measured retrospectively in 21 subjects with RP. The Wilcoxon matched-pairs signed-rank test was used for statistical comparisons of biomarker levels in active and inactive phases of RP. RESULTS: Only the serum level of COMP was significantly increased during disease flares. Steroids did not alter the serum cartilage-related biomarker levels. However, during the active phase, C2C levels were significantly higher in steroid treated patients compared with non-steroid treated patients. CONCLUSIONS: This study suggests that serum COMP level may be useful for monitoring disease activity of RP. Further prospective studies are required to confirm this result.

Biomarkers associated with clinical phenotypes of hand osteoarthritis in a large multigenerational family: the CARRIAGE family study.

OBJECTIVE: To evaluate biological markers as potential quantitative traits of clinical osteoarthritis (OA) in a large multigenerational family in the Carolinas of the USA known as the CARRIAGE (CARolinas Region Interaction of Aging, Genes and Environment) family. METHODS: During a series of three family reunions over 6 years, we ascertained 365 family members. We performed clinical hand examinations (n=287), and obtained sera (n=278) for seven OA-related biomarkers [type IIA collagen N-propeptide (PIIANP), type II procollagen carboxy-propeptide (CPII), neoepitope from cleavage of CII (C(2)C), cartilage oligomeric matrix protein (COMP), hyaluronan (HA), high-sensitive C-reactive protein (hs-CRP), and glycated serum protein (GSP)]. Three hand OA definitions were evaluated--clinical ACR (American College of Rheumatology) and GOGO (Genetics of Generalized OA) criteria, and any single hand joint involvement. Non-hand OA was defined as a negative hand examination for OA but varying prevalence of joint symptoms; the control group was defined as having neither symptoms nor evidence for clinical hand OA. RESULTS: Mean lnHA, lnCOMP, and lnhs-CRP were significantly higher in the hand OA group, compared with the non-hand OA or control group. Adjusted for age and sex, mean lnPIIANP (a collagen II synthesis marker) was significantly lower in the hand OA group compared with the other groups. Among those without clinical hand OA, GSP was associated with hand joint symptoms. CONCLUSIONS: This is the first report, to our knowledge, showing an association of OA biomarkers and hand OA based on physical examination alone. Analyses using these biomarkers as quantitative traits could reveal novel genetic loci and facilitate exploration of the genetic susceptibility to OA.

Measurement of synovial fluid volume using urea.

OBJECTIVE: To examine the utility of using urea concentrations for determining Synovial Fluid (SF) joint volume in effused and non-effused joints. METHODS: Knee joint SF was aspirated from 159 human study participants with symptomatic osteoarthritis of at least one knee either directly (165 knees) or by lavage (110 knees). Serum was obtained immediately prior to SF aspiration. Participants were asked to rate individual knee pain, aching or stiffness. SF and serum urea levels were determined using a specific enzymatic method run on an automated CMA600 analyzer. Cell counts were performed on direct SF aspirates when volume permitted. The formula for calculating SF joint volume was as follows: V(j)=C(D)(V(I))/(C-C(D)) with V(j)=volume of SF in entire joint, C(D)=concentration of urea in diluted (lavage) SF, V(I)=volume of saline injected into joint, and C=concentration of urea in undiluted (neat) SF derived below where C=0.897(C(S)) and C(s)=concentration of urea in serum. RESULTS: There was an excellent correlation (r(2)=0.8588) between SF and serum urea in the direct aspirates with a ratio of 0.897 (SF/serum). Neither urea levels nor the SF/serum ratio showed any correlation with Kellgren Lawrence (KL) grade, or cell count. While urea levels increased with age there was no change in the ratio. Intraarticular SF volumes calculated for the lavaged knees ranged from 0.555 to 71.71ml with a median volume of 3.048ml. There was no correlation of SF volume to KL grade but there was a positive correlation (P=0.001) between SF volume and self-reported individual knee pain. CONCLUSION: Our urea results for direct aspirates indicate an equilibrium state between serum and SF with regard to the water fraction. This equilibrium exists regardless of disease status (KL grade), inflammation (cell count), or age, making it possible to calculate intraarticular volume of lavaged joints based upon this urea method. Most of the joint volumes we calculated fell within the previously reported range for normal knees of 0.5-4.0ml. The positive correlation between SF volume and knee symptoms reinforces the clinical utility of this method for quantifying SF volume.

Interpretation of serum C-reactive protein (CRP) levels for cardiovascular disease risk is complicated by race, pulmonary disease, body mass index, gender, and osteoarthritis.

OBJECTIVE: High-sensitivity C-reactive protein (hsCRP) in serum is used as a marker of risk for cardiovascular disease (CVD); however CRP is a non-specific acute phase reactant. We evaluated the association between hsCRP concentrations and the most common form of arthritis, osteoarthritis (OA), and assessed the applicability of hsCRP for CVD risk prediction. METHODS: Participants (n=662) were selected from the population-based Johnston County Osteoarthritis Project, using stratified simple random sampling to achieve balance according to radiographic knee OA status, ethnic group, gender, and age group. The presence and severity of knee and hip OA were determined radiographically. CVD risk was estimated by hsCRP concentration and independently with the Framingham risk algorithm. RESULTS: Serum natural log-transformed hsCRP (ln hsCRP) was higher in African-Americans (P<0.0001) and women (P<0.0001), was higher in participants who had chronic pulmonary disease (P=0.01), hypertension (P<0.0001), or used pain medications (P=0.004), and correlated with body mass index (BMI) (r=0.40, P<0.0001) and waist circumference (r=0.33, P<0.0001), but not with age, CVD, or current smoking. Ln hsCRP was strongly positively associated with all definitions of radiographic OA (rOA; P<0.0001), but this association was not independent of BMI. Although 183 participants reported no CVD and were classified as low risk by the Framingham CVD score, 61% of them were classified as moderate or high risk for CVD using hsCRP; this proportion designated high risk for CVD on the basis of hsCRP consisted primarily of women (P<0.05) and individuals with OA (P<0.01). CONCLUSIONS: The pathogenic significance of hsCRP elevations in this subgroup is unclear. Serum hsCRP for predicting risk of CVD is confounded by obesity, ethnicity, gender and comorbidities.

Diurnal variation of serum and urine biomarkers in patients with radiographic knee osteoarthritis.

OBJECTIVE: To evaluate diurnal variation of biomarkers in subjects with osteoarthritis (OA) of the knee. METHODS: Twenty subjects with radiographic knee OA were admitted to the General Clinical Research Center of Duke University for an overnight stay to undergo serial blood and urine sampling. Biomarkers measured included serum hyaluronan (HA), cartilage oligomeric matrix protein (COMP), keratan sulfate (KS-5D4), aggrecan neoepitope (CS846), high-sensitivity C-reactive protein (hsCRP), osteocalcin, transforming growth factor beta1 (TGFbeta1), and type II collagen (CII)-related epitopes (neoepitope from cleavage of CII [C2C], carboxy-terminus of three-quarter peptide from cleavage of CI and CII [C1,2C], and type II procollagen carboxy-propeptide [CPII] in serum, and C-terminal telopeptides of CII [CTX-II] and C2C in urine). RESULTS: Levels of serum HA, COMP, KS-5D4, and TGFbeta1 increased significantly from T0 (before arising from bed) to T1 (1 hour after arising). More diurnal variation in HA was observed in patients with higher daily mean HA concentrations. CPII increased significantly from T0 to T2 (4 hours after arising). Urinary concentrations of CTX-II were also found to vary with morning activity, decreasing significantly from T0 to T2. Urinary C2C concentrations increased significantly from T0 until T3 (early evening). No diurnal variations in CS846, hsCRP, osteocalcin, serum C2C, or C1,2C were observed. Six biomarkers (serum C2C, C1,2C, COMP, KS-5D4, TGFbeta1, and urinary CTX-II) were associated with radiographic knee OA (expressed as the sum of Kellgren/Lawrence radiographic severity grades), with the strongest correlations observed with measurements obtained at later time points (either T2 or T3). CONCLUSION: Our study results suggest that serum and urine sampling for HA, COMP, KS-5D4, TGFbeta1, CPII, urinary CTX-II, and urinary C2C should be standardized in future OA clinical trials. Serum and urine sampling at late midday time points may be the optimal approach for OA studies, although this result should be validated in a larger cohort.

The influence of mechanical compression on the induction of osteoarthritis-related biomarkers in articular cartilage explants.

OBJECTIVE: Macromolecules of the articular cartilage extracellular matrix released into synovial fluid, blood, or urine can serve as potentially useful biomarkers of the severity of osteoarthritis (OA). Biomechanical factors play an important role in OA pathogenesis, yet their influence on biomarker production is not well understood. The goal of this study was to examine the hypothesis that dynamic mechanical stress influences the release of these biomarkers from articular cartilage. METHODS: Explants of porcine cartilage were subjected to dynamic compression at 0.5 Hz for 24h at stresses ranging from 0.006 to 0.1 MPa. The concentrations of cartilage oligomeric matrix protein (COMP), keratan sulfate (KS measured as the 5 D 4 epitope), total sulfated glycosaminoglycan (S-GAG), and the KS (keratanase-digestible) and chondroitin sulfate (CS) (chondroitinase-digestible) fractions of S-GAG were measured. Radiolabel incorporation was used to determine the rates of proteoglycan and protein synthesis. RESULTS: The magnitudes of mechanical stress applied in this study induced nominal tissue strains of 4-23%, consistent with a range of physiological to hyperphysiologic strains measured in situ. COMP release increased in proportion to the magnitude of dynamic mechanical stress, while KS, CS and total S-GAG release increased in a bimodal pattern with increasing stress. Protein and proteoglycan synthesis were significantly decreased at the highest level of stress. CONCLUSION: Mechanical stress differentially regulates the turnover of distinct pools of cartilage macromolecules. These findings indicate that mechanical factors, independent of exogenous cytokines or other stimulatory factors, can influence the production and release of OA-related biomarkers from articular cartilage.

Antigen based therapies to prevent diabetes in NOD mice.

Interventional approaches that have been successful in delaying insulin-dependent diabetes mellitus (IDDM) using antigen-based immunotherapies include parenteral immunization. It has potential for clinical application provided that effective adjuvants suitable for human use can be found. We have previously shown that immunization with insulin and insulin B chain but not A chain in incomplete Freund's adjuvant (IFA) prevented diabetes by reducing IFN-gamma mRNA in the insulitis lesions. In this paper we show that the insulin B chain peptide (p9-23) contain the most protective epitope. Immunization with selected GAD peptides was ineffective. Immunization with B chain but not A chain using alum as adjuvant delayed diabetes onset (P = 0.012), whereas administration of alum alone was not protective. When Diphtheria-Tetanus toxoid-Acellular Pertussis (DTP) vaccine was used as the adjuvant vehicle, DTP itself induced significant protection (P < 0.003) which was associated with a Th2-like cytokine producing insulitis profile, IL-4 driven IgG1 antibody responses to insulin, GAD in the periphery and an augmentation of the autoimmune response to GAD. The anti-diabetic effect of DTP was enhanced when given with insulin B chain. These results encourage consideration of an approach using alum/DTP and insulin B chain immunization in clinical trials.

Counter-regulatory hormone responses to insulin-induced acute hypoglycemia in hypopituitary patients.

Patients with hypopituitarism are predisposed to fasting hypoglycemia and are considered unusually sensitive to insulin-induced acute hypoglycemia. However, whether impaired response of counter-regulatory hormones, such as glucagon, epinephrine (E), and nor-epinephrine (NE) contribute to the susceptibility to acute hypoglycemia in hypopituitary patients has not been systematically evaluated. Therefore, we compared counter-regulatory hormone responses to insulin-induced acute hypoglycemia in 9 patients with hypopituitarism who were off hormone replacement therapy and 13 normal healthy subjects. All subjects received aa prime-continuous intravenous infusion of insulin (0.1 Unit/kg body weight.h) till plasma glucose declined to less than 2.5 mmol/l or occurrence of hypoglycemic symptoms. All normal subjects and 7 out of 9 hypopituitary patients recovered spontaneously from hypoglycemia. Two hypopituitary patients with hypothalamic pathology however needed intravenous glucose, glucagon and hydrocortisone to assist recovery from hypoglycemia. Overall, patients with hypopituitarism showed a slower rate of recovery of plasma glucose after hypoglycemia than normal subjects (0.78 +/- 0.33 mmol/l.h vs. 1.72 +/- 0.15 mmol/l.h, respectively; p = 0.02). The responses of key counter-regulatory hormones, glucagon, E and NE, to hypoglycemia however were essentially similar in both the groups. We conclude that the lack of cortisol (secondary to ACTH deficiency) and GH in hypopituitary patients may be primarily responsible for the slow recovery of plasma glucose after acute hypoglycemia; and plasma glucagon, E, and NE responses are not impaired.

Chemiluminescence immunoassay of aldosterone in serum.

A chemiluminescence immunoassay (CLI) for the direct measurement of aldosterone in serum was developed with aminobutylethyl isoluminol (ABEI) as the label. In this competitive assay the samples are incubated with sample, antibody, aldosterone-carboxymethyl oxime-ABEI, and paramagnetic particles coated with second antibody. After magnetic separation and washing, the samples are incubated with 200 microL of NaOH (2 mol/L) at 60 degrees C for 30 min. In the luminometer the chemiluminescence is produced by the serial injection of 150 microL each of microperoxidase and H2O2 solutions. Comparison of results with an RIA method showed excellent agreement: CLI = 1.001 RIA + 0.020 (r = 0.99, n = 93). The method is simple and avoids the hazards and costs associated with isotopic waste. The label has a shelf life of at least two years.

Chemiluminescence immunoassay of cyclosporine in whole blood.

We describe a chemiluminescent immunoassay (CLI) for measuring cyclosporine in whole blood. Its sensitivity and accuracy are comparable with those of an RIA method that makes use of the same specific monoclonal antibody. The comparison with the RIA method was excellent: y(RIA) = x(CLI) + 11.24 (r = 0.99). In our procedure the samples are incubated with cyclosporin C-hemisuccinate-aminobutyl-N-ethylisoluminol, antibody, and paramagnetic particles coated with second antibody. After magnetic separation and washing, the samples are incubated with 200 microL of NaOH (2 mol/L) at 60 degrees C for 30 min. The chemiluminescence generated by automated serial injections of solutions of microperoxidase and dilute (2 mL/L) H2O2 is measured for 5 s. The data are processed by using a spline fit of log B/Bo log conversion. This method is easy to perform and avoids the hazards and costs associated with isotopic waste disposal. The label is stable for at least three years.

A commercially available assay kit for determining aldosterone concentration in non-extracted rat serum.

A commercially available assay for aldosterone that requires no extraction was found to be valid without modification for rat serum. The assay was performed in duplicate on as little as 0.5 cc of rat serum and gave linear results from 5 ng/dl to 500 ng/dl, (i.e., the physiological range of the rat). Samples above 150 ng/dl had to be diluted in this assay. The cost per sample if performed in duplicate and in batches was approximately one dollar. We found that this assay, which was developed for and is marketed for determining aldosterone concentration in human samples, offered a cost effective assay for aldosterone determination in the rat.

Laboratory evaluation of the Boehringer Mannheim "Diagnostic M" automated discrete analyzer.

We evaluated a new multiple-channel chemistry analyzer, the Boehringer Mannheim "Diagnostic M." This instrument can perform 25 tests at the rate of 120 1.3-mL serum samples per hour. The instrument may be run in either a profile mode or single-test mode. In the single-test mode only the necessary reagent is pumped. the instrument is computer controlled. We compared it with the Technicon SMAC, SMA 12/60, and SMA 6/60. It demonstrated excellent precision, linearity, lack of interference, ease of operation, and satisfactory comparison with values obtained by the Technicon methods.