Background-Risk stratification in troponin (cTn)-negative acute coronary syndrome (ACS) remains a clinical challenge.We investigated the predictive value of circulating pregnancy-associated plasma protein A (PAPP-A), a novel marker of atherosclerotic plaque activity, in these patients. Methods and Results-Two hundred consecutive hospitalized ACS patients were included, of whom 136 (69 men and 67 women; meanϮSD age, 66Ϯ16 years) remained cTnI-negative for up to 24 hours. PAPP-A was measured at admission, 6 to 12 hours, and 24 hours. During 6-month follow-up, 26 (19.1%) of the cTnI-negative patients reached a primary end point (cardiovascular death, myocardial infarction, or revascularization). At a cutoff level of 2.9 mIU/L, elevated PAPP-A was an independent predictor of adverse outcome (adjusted risk ratio [RR], 4.6; 95% confidence interval, 1.8 to 11.8; Pϭ0.002). Another independent predictor was admission CRP Ͼ2.0 mg/L (RR, 2.6; Pϭ0.03). Conclusions-Measurement of plasma PAPP-A, a zinc-binding matrix metalloproteinase, is a strong independent predictor of ischemic cardiac events and need of revascularization in patients who present with suspected myocardial infarction but remain troponin negative. (Circulation. 2003;108:1924-1926.)

Background: Reverse transcription-PCR (RT-PCR) assays have been used for analysis of circulating tumor cells (CTCs), but their clinical value has yet to be established. We assessed men with localized prostate cancer or castration-refractory prostate cancer (CRPC) for CTCs via real-time RT-PCR assays for KLK3 [kallikrein-related peptidase 3; i.e., prostate-specific antigen (PSA)] and KLK2 mRNAs. We also assessed the association of CTCs with disease characteristics and survival. Methods: KLK3, KLK2, and PSCA (prostate stem cell antigen) mRNAs were measured by standardized, quantitative real-time RT-PCR assays in blood samples from 180 localized-disease patients, 76 metastatic CRPC patients, and 19 healthy volunteers. CRPC samples were also tested for CTCs by an immunomagnetic separation system (CellSearch™; Veridex) approved for clinical use. Results: All healthy volunteers were negative for KLK mRNAs. Results of tests for KLK3 or KLK2 mRNAs were positive (≥80 mRNAs/mL blood) in 37 patients (49%) with CRPC but in only 15 patients (8%) with localized cancer. RT-PCR and CellSearch CTC results were strongly concordant (80%–85%) and correlated (Kendall τ, 0.60–0.68). Among CRPC patients, KLK mRNAs and CellSearch CTCs were closely associated with clinical evidence of bone metastases and with survival but were only modestly correlated with serum PSA concentrations. PSCA mRNA was detected in only 7 CRPC patients (10%) and was associated with a positive KLK mRNA status. Conclusions: Real-time RT-PCR assays of KLK mRNAs are highly concordant with CellSearch CTC results in patients with CRPC. KLK2/3-expressing CTCs are common in men with CRPC and bone metastases but are rare in patients with metastases diagnosed only in soft tissues and patients with localized cancer.

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Homocysteine has been suggested to be a risk factor for fracture, but the causal relationship is not clear. In 996 women from the OPRA study, high homocysteine level was associated with high bone marker levels and low BMD at baseline. During a mean 7-year follow-up, high homocysteine level was associated with mortality, but no clear association to fracture risk existed.Introduction: Recently, the association between high serum homocysteine (Hcy) levels and an increased risk of fracture has been described. Materials and Methods: Hcy levels were measured at baseline in 996 women, all 75 years old. Vitamin B 12 , folate, serum cross-linking telopeptide of type I collagen (CTX), serum TRACP5b, serum osteocalcin, urine deoxypyridinoline, PTH, areal BMD (aBMD), calcaneal quantitative ultrasound (QUS), and physical performance were assessed at baseline. Fractures and mortality were recorded during a mean follow-up of 7.0 years. Results: Bone marker levels were higher in women with Hcy in the highest quartile compared with all other women (p < 0.05). The most evident correlation between Hcy and a bone marker was seen with CTX (r ‫ס‬ 0.19, p < 0.001). aBMD (hip) was 4% lower, QUS was up to 2% lower, and gait speed was 11% slower among women with Hcy in the highest quartile compared with the other women (p < 0.05). During the follow-up, 267 women sustained at least one low-energy fracture (including 69 hip fractures). When women in the highest Hcy quartile were compared with all other women, the hazard ratios (HRs) for sustaining any type of fracture was 1.18 (95% CI, 0.89-1.36) and for hip fracture was 1.50 (95% CI, 0.91-1.94). For the same group of women, the mortality risk was 2.16 (95% CI, 1.58-2.55). Adjustments for confounders did not substantially change these associations. Adjustment for PTH increased the HR for hip fracture to 1.67 (95% CI, 1.01-2.17). Low vitamin B 12 or folate was not associated with increased fracture risk or mortality. Conclusions: High Hcy levels were associated with higher bone turnover, poor physical performance, and lower BMD. There was no clear association to fracture risk. The increased mortality among women with high Hcy levels indicates that a high Hcy level may be a marker of frailty.

Background: Cardiac troponin I (cTnI) is a sensitive marker of cardiac injury, but cTnI assays, like other immunoassays, are susceptible to interferences. We evaluated the presence of interfering substances by measuring the recovery of cTnI added to samples from volunteers and from patients with acute coronary syndromes (ACS). Methods: We added a ternary complex of human cardiac troponin (30 -500 g/L) or cTnI from serum to samples from healthy volunteers and ACS patients. We measured cTnI with a two-site sandwich time-resolved immunofluorometric assay using two antibodies against epitopes in the central stable part of cTnI. We also analyzed 108 heparin-plasma samples from 16 ACS patients with this assay, with an assay based on four antibodies, and with two commercial cTnI assays, Ax-SYM and ACS:180. Results: In samples from both healthy persons and ACS patients, recoveries for our assay were 1-167% (range). Recoveries were increased by addition of an antibody with an epitope in the N-terminal region of cTnI to the solid phase and an antibody with an epitope in the C-terminal region as a second detection antibody. In 2 of 16 patients with ACS, normal cTnI concentrations found when measured with the original assay demonstrated clinically abnormal (up to 10-fold higher) results with the additional N-and C-terminal antibodies in the early phase of infarction. Both commercial cTnI assays also

BackgroundV-betaLH is a common genetic variant of LH caused by two polymorphic base changes in the beta subunit gene, altering the amino acid sequence (Trp8Arg and Ile15Thr). In a previous-preliminary trial performed in women undergoing IVF, it was demonstrated that carriers of v-betaLH show sub-optimal ovarian response to a standard long GnRH-agonist down -regulation protocol when stimulated with pure recombinant FSH (r-hFSH). The aim of this study was to confirm the hypothesis that women with v-betaLH display hypo-sensitivity to exogenous FSH in a larger IVF population and to explore the frequency of this variant in a Danish female population.MethodsIn the present study, the effect of v-betaLH was retrospectively investigated in a larger series of women undergoing controlled ovarian stimulation (COS) and, for the first time, in a Danish IVF population. A total of 220 normogonadotrophic women following a long GnRH-agonist down-regulation protocol received an individualized dose of r-hFSH (100 IU and 375 IU s.c. daily) according to antral follicle count, baseline FSH, body mass index and age. The LH genotype was assessed in all patients by immunofluorometric assay.ResultsV-betaLH was present in 11% of patients, whereas the allelic frequency was 12%. The study population was divided into two groups according to their LH genotype. Group A consisted of 196 wt/wt women. Group B included 24 individuals with v-betaLH (21 heterozygous and 3 homozygous). No statistically significant differences in the mean number of oocytes retrieved, fertilization rate and pregnancy rate per cycle were observed between groups. However, Group B received a significantly higher cumulative-dose of r-hFSH than Group A (2435.86 +/− 932.8 IU versus 1959.8 +/− 736.45 p = 0.048). When one-way ANOVA in a within design was applied, the LH genotype had a statistically significant effect (p < 0.01) on the cumulative dose of r-hFSH, showing a progressive increase from wt/wt (1959.8 +/− 736.45 IU) to v-betaLH hetero- (2267.5 +/− 824.3) and homozygotic women (3558.3 +/− 970.9).ConclusionsThese results confirm that carriers exhibit hypo-sensitivity to exogenous FSH during COS, documenting that the frequency of v-betaLH in Denmark is similar to a number of European countries.

We have analyzed by different immunological methods the proteolytic degradation of cardiac troponin I (cTnI) in human necrotic tissue and in serum. cTnI is susceptible to proteolysis, and its degradation leads to the appearance of a wide diversity of proteolytic peptides with different stabilities. N- and C-terminal regions were rapidly cleaved by proteases, whereas the fragment located between residues 30 and 110 demonstrated substantially higher stability, possibly because of its protection by TnC. We conclude that antibodies selected for cTnI sandwich immunoassays should preferentially recognize epitopes located in the region resistant to proteolysis. Such an approach can be helpful for a much needed standardization of cTnI immunoassays and can improve the sensitivity and reproducibility of cTnI assays.

Fourteen monoclonal antibodies (mAbs) against human cardiac troponin I (cTnI) were generated by commonly used experimental techniques. All these antibodies, as well as antibody 414 (HyTest), were specific for human cTnI. Fifteen antibodies thus obtained were tested in a sandwich cTnI immunofluorescence assay (altogether 196 combinations). Ten pairs giving the highest sensitivity were selected for further investigation. The effect of TnI–TnC complex formation on antibody interaction with antigen was analyzed. The formation of TnI–TnC complex results in a significant decrease of the interaction of mAbs with TnI for seven of 10 analyzed pairs of antibodies. Using two pairs of cTnI-specific mAbs, one that recognized only free cTnI but not cTnI complexed with cTnC, and another that could be used for measurement of total cTnI (free cTnI and cTnI in complex with cTnC), we demonstrated that the main part of cTnI in serum collected from acute myocardial infarction patients is presented in the complex form. We concluded that effective and reliable immunological detection of TnI is possible only when antibodies used for assay development recognize both free TnI and TnI complexed with other troponin components.