Multi-center evaluation of analytical performance of the microparticle enzyme immunoassay for sirolimus.

OBJECTIVES: This study evaluated the analytical characteristics of the new Abbott microparticle enzyme immunoassay (MEIA) for sirolimus. DESIGN AND METHODS: The protocol consisted of nine sections: evaluation of antibody specificity, linearity, detection limit, quantification limit, endogenous interferents, exogenous interferents, precision, proficiency testing panel, and method comparison. RESULTS: The mean analytical detection limit was 0.68 microg/L. The sirolimus concentration corresponding to a total CV of 20% was 1.5 microg/L. Linearity of response was demonstrated across the dynamic range of the assay. Total precision (CVs) at QC control levels from 5 to 22 microg/L ranged from 5.7 to 12.6%. Assay standardization was found to be in good agreement with LC/MS/MS as compared with target values for spiked sirolimus proficiency samples from an international sirolimus proficiency testing program. Good correlations (R values) of the immunoassay were observed in comparisons to LC/MS/MS. R values tended to be lower in comparisons with LC/UV methods. Across both LC-based methods and all study sites, there was approximately 25% overall positive slope bias due to cross reactivity of the MEIA antibody to metabolites of sirolimus. The assay cross-reactivity to metabolites of sirolimus parent drug ranged from 6 to 63%. Assay interferences were minimal with the exception of hematocrit, which presented a negative relationship to measured sirolimus concentration. CONCLUSIONS: The MEIA demonstrated acceptable analytical characteristics for use for routine monitoring of sirolimus immunosuppressive therapy, and is a viable alternative to HPLC-based methods for sirolimus monitoring.

WITHDRAWN: Multi-center evaluation of analytical performance of the microparticle enzyme immunoassay for sirolimus.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Clin. Biochem. 39 (2006) 378-386, doi:10.1016/j.clinbiochem.2006.01.017. The duplicate article has therefore been withdrawn. This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The Publisher apologizes for any inconvenience this may cause.

Effects of angiotensin II blockade on nitric oxide blood levels in IgA nephropathy.

BACKGROUND: The effects of renin-angiotensin system blockade on nitric oxide (NO), especially in pathological conditions, are far from being established. The influence of kinins and angiotensin type 2 receptor are largely speculative and based mainly on animal studies. This study was aimed to address these aspects in humans. METHODS: Eight IgA nephropathy patients with documented clinical and histological indicators of poor prognosis were given 50 mg of losartan, 10 mg of enalapril, and 40 mg of the NO donor isosorbide 5 mononitrate (as a control of NO generation) in randomized order for 7 days each. Treatment periods were separated by washout periods of 7 days each. Laboratory investigations were performed before and after each study period. Seven healthy controls received losartan and enalapril according to the same study design. RESULTS: Glomerular filtration rate remained stable while effective renal plasma flow increased with each treatment (P<0.05). Under losartan and enalapril, filtration fraction fell (P=0.02), plasma renin activity increased (P<0.05) and urinary aldosterone concentration decreased (P=0.02). Angiotensin-converting enzyme activity was reduced to the limit of detection under enalapril (P<0.001). Blood NO, detected as nitrosylhaemoglobin by a recently developed technique of spin-trap electron paramagnetic resonance, increased significantly, as expected, during treatment with isosorbide 5 mononitrate (P=0.01), with enalapril (P<0.05), and also with losartan (P<0.05). Unlike losartan, enalapril significantly reduced albuminuria (P=0.01) in this short-term period. In the seven healthy controls, neither enalapril nor losartan were able to increase blood NO levels significantly. CONCLUSIONS: Blood levels of nitrosylhaemoglobin, a surrogate marker of NO, increased under blockade of the renin-angiotensin system in patients with IgA nephropathy, but not in healthy volunteers. This increase could contribute to changes of effective renal plasma flow in renal disease states.

Isosorbide 5 mononitrate administration increases nitric oxide blood levels and reduces proteinuria in IgA glomerulonephritis patients with abnormal urinary endothelin/cyclic GMP ratio.

An endothelin urinary hyperexcretion, which is not counterbalanced by an adequate increase in cGMP biosynthesis, was previously detected in some patients with IgA Nephropathy (IgAN). Since this imbalance might potentiate local ET1-mediated hemodynamics effects, 9 IgAN patients with an increased (> or = 0.1) urinary ET1/cGMP ratio (group 1) and 5 IgAN patients with comparable renal function and reduced ET1/cGMP ratio (group 2) were given standard doses of isosorbide 5 mononitrate (as a nitric oxide source). Blood nitric oxide (NO) levels, as detected by electron paramagnetic resonance, significantly increased after isosorbide administration (p < 0.01) and decreased after drug discontinuation in both groups. Nitric oxide levels were significantly related with those of the effective renal plasma flow (p < 0.02), but not with the glomerular filtration rate. Proteinuria levels significantly decreased after drug administration (p < 0.009) in group 1 and returned to baseline levels thereafter, except two cases showing persisting low levels. Values of filtration fraction in the same group decreased after iso5M administration (p < 0.02 compared to basal levels). These results may possibly be related to the counterbalancing effects of nitric oxide on endothelin-mediated mesangial contraction.

Urinary endothelin in glomerulonephritis patients with normal renal function.

The vasoconstrictor peptide endothelin-1 (ET1) has only recently been characterized and its effects are at present largely speculative. It has been hypothesized that ET1 acts on mesangial cells to cause vasoactive changes which might ultimately contribute to the development of glomerulosclerosis. Opposite to ET1, nitric oxide (NO) inhibits mesangial cell contraction and proliferation. NO activates soluble guanylic acid cyclase and the final product, cyclic GMP (cGMP), has been recently used as a marker of NO action. Urinary levels of ET1 and cGMP were detected in 58 patients with biopsy-proven glomerulonephritis (GN), including 36 IgA nephropathy (IgAGN), 30 with normal and 6 with impaired renal function, 10 patients with non-IgA mesangial GN and 12 pts with membranous GN (MGN) with normal renal function. Compared to normal controls (0.019 +/- 0.006 ng/min), urine ET1 levels were significantly higher in patients with normal renal function having IgAGN (0.035 +/- 0.017, p < 0.01), MGN (0.028 +/- 0.013, p < 0.05), non-IgA mesangial GN (0.027 +/- 0.012, p < 0.05) and those with IgAGN and renal failure (0.032 +/- 0.011, p < 0.01). However no difference was found between MGN patients and normals by deleting MGN cases with mild to moderate mesangial proliferation. The mean value of urinary cGMP in IgAGN patients with renal failure (0.186 +/- 0.117 nmol/min) was lower (p < 0.05) than that of each group with normal renal function (IgAGN: 0.378 +/- 0.010 nM/min; MGN: 0.338 +/- 0.064 nmol/min, non-IgAGN: 0.436 +/- 0.168 nmol/min). The same significant differences were obtained by correcting cGMP values for creatinine urinary excretion. Urinary ET/cGMP ratio (assumed as an index of the relative balance between vasoconstrictor and vasorelaxing factors) was found to be higher than normal (0.570 +/- 0.010 ng/nmol) both in IgAGN patients with normal renal function (0.103 +/- 0.064 ng/mol, p < 0.05), and in those with renal failure (0.203 +/- 0.108 ng/nmol, p < 0.02). Urinary cGMP values were not related to plasma levels of atrial natriuretic peptide (ANP). These data show that hyperexcretion of ET1 occurs in a number of patients with mesangial proliferative GN. In some of them, mainly those with established glomerular damage, the local production of ET1 is not counter-balanced by adequate cGMP biosynthesis.

Estimation of chromium-51 ethylene diamine tetra-acetic acid plasma clearance: a comparative assessment of simplified techniques.

Chromium-51 ethylene diamine tetra-acetic acid (51Cr-EDTA) total plasma clearance was evaluated using a multi-sample method (i.e. 12 blood samples) as the reference compared with several simplified methods which necessitated only one or few blood samples. The following 5 methods were evaluated: terminal slope-intercept method with 3 blood samples, simplified method of Bröchner-Mortensen and 3 single-sample methods (Constable, Christensen and Groth, Tauxe). Linear regression analysis was performed. Standard error of estimate, bias and imprecision of different methods were evaluated. For 51Cr-EDTA total plasma clearance greater than 30 ml.min-1, the results which most approximated the reference source were obtained by the Christensen and Groth method at a sampling time of 300 min (inaccuracy of 4.9%). For clearances between 10 and 30 ml.min-1, single-sample methods failed to give reliable results. Terminal slope-intercept and Bröchner-Mortensen methods were better, with inaccuracies of 17.7% and 16.9%, respectively. Although sampling times at 180, 240 and 300 min are time-consuming for patients, 51Cr-EDTA total plasma clearance can be accurately calculated for values greater than 10 ml.min-1 using the Bröchner-Mortensen method. In patients with clearance greater than 30 ml.min-1, single-sample techniques provide a good alternative to the multi-sample method; the choice of the method to be used depends on the degree of accuracy required.

Increased serum neopterin concentration as indicator of disease severity and poor survival in multiple myeloma.

In this study we investigated serum neopterin levels in 73 multiple myeloma (MM) patients (63 determinations at diagnosis, 58 in remission, and 35 at relapse), in 56 monoclonal gammopathies of undetermined significance (MGUS), and in 70 normal controls. Median neopterin level was 5.3 nmol/l in normal controls, 6.8 nmol/l in MGUS, and 10.7 nmol/l in MM patients. In comparison to healthy subjects, significantly higher levels were observed in MM patients (p less than 0.0001). A statistical difference was observed between MGUS and MM patients at diagnosis (p less than 0.007). Compared to diagnosis, a further increase was noticed during relapse, suggesting a correlation between neopterin and disease activity. The prognostic significance of raised neopterin levels was confirmed by a survival analysis. Median survival for patients with high values was 20 months, whereas it was 63.9 months for those with low values (log-rank test p less than 0.003). Serum neopterin concentrations also correlated to beta 2 microglobulin levels and the percentage of CD38+ circulating lymphocytes, indicating a link between neopterin and other myeloma prognostic factors.

The fate of aggregated immunoglobulin A injected in IgA nephropathy patients and healthy controls.

Organ uptake of IgA-containing immunologically active material was studied in humans by intravenous (IV) injection of 131I-labeled heat-aggregated human secretory IgA (HAS-IgA) in nine patients affected by primary IgA nephropathy and 10 normal volunteers. Aggregated secretory IgA was found to be removed almost exclusively by the liver. The peak activity in liver was reached at 21.1 minutes (range, 18 to 26 minutes) in patients and 19 minutes (range, 14 to 22 minutes) in controls. The rate of increase of liver radioactivity was found to be significantly slower in patients (with a mean slope of 5.0; range, 3.4 to 7.1 v 7.6, 5.6 to 11.4; P less than 0.02). The mean liver to precordium ratio at the peak time was significantly lower in patients (mean value, 2.3; range, 1.9 to 3.1) compared with controls (mean value, 3.3; range, 2.4 to 4.0) (P less than 0.02). These data confirm the pivotal role of the liver in the removal of aggregated IgA in humans and the defective clearance capacity of this test probe in IgA nephropathy patients.

[Tag 72: a new tumor-associated antigen identified by a monoclonal antibody]. / Tag 72: un nuovo antigene tumore associato riconosciuto da un anticorpo monoclonale.

In order to evaluate the usefulness of Tag 72--tumor associated antigen assay--in gastroenterology, we have studied with Ca 72-4 radioimmunoassay (Centocor) 551 patients suffering benign (233) and neoplastic (318) gastrointestinal diseases and 205 normal controls. The cut-off point was fixed at 6 U/ml. Only in gastric cancers, the Tag 72 assay, with the proposed method, provide additional information in this pathology (sensitivity 30%, specificity 98.7%). The most striking observation to be made from the current study is a poor sensitivity of the test for gastrointestinal cancers, but rather the excellent specificity of the Ca 72-4 IRMA with respect to benign gastrointestinal diseases. The sensitivity of Ca 72-4 assay, vs Ca 19-9 and CEA, for the same diseases, is less, but specificity is better.

Significance of CA 72.4 serum levels in gastrointestinal diseases.

In order to evaluate the usefulness of Ca 72.4 tumor associated antigen assay in gastrointestinal diseases, we have studied 751 patients suffering from benign (376) and neoplastic (375) digestive diseases and 305 normal controls. The cut-off point was fixed at 6 U/ml. The Ca 72.4 assay, with the proposed method, provides additional information only in gastric cancers; the positivity of the marker in gastric neoplasms is 38.4% and the specificity vs gastric ulcers and atrophic gastritis is 99%. In six patients with gastric cancer, the Ca 72.4 is the only positive test. The most striking observation to be made from the current study is a no good sensitivity of the marker for gastrointestinal cancers (29.6% vs 35.7 and 37.6% for CEA and Ca 19-9 respectively), but rather the excellent specificity of the Ca 72.4 immunoassay with respect to being gastrointestinal diseases (98.7%), vs values of specificity for CEA and Ca 19-9 of 94 and 92%. In conclusion, the high specificity of this marker for gastrointestinal neoplasms may be very interesting in follow-up studies. In fact, an elevation of serum levels of Ca 72.4 should always be taken seriously.

[Determination of tumor-associated trypsin inhibitor (TATI) in subjects with gastrointestinal diseases. Preliminary data]. / Dosaggio del tumor associated trypsin inhibitor (TATI) in soggetti con malattie gastrointestinali. Dati preliminari.

Tumor-associated trypsin inhibitor (TATI) is a 6 K dalton protease inhibitor, that was isolated from urine of a patient with ovarian cancer. In our experience, mean serum level of TATI in healthy subjects (n. 120), is 13 micrograms/l (range 5.1-42 micrograms/l). The cut-off point is established in 32 micrograms/l (mean +/- 3 SD). We have examined 357 patients with gastrointestinal diseases: 98 gastric cancer, 50 colon cancers, 52 pancreatic cancers, 32 chronic pancreatitis, 38 IBD, 28 colon polyps, 40 gastric ulcers and 25 non-neoplastic biliary tree diseases. TATI may be a good tumor marker only in gastric cancer. Elevated levels of TATI also occur in obstructive hepatobiliary disease and active pancreatitis or IBD.

Changes induced by fasting and cycloheximide in the vacuolar apparatus of rat hepatocytes. A morphometric investigation.

An increase in the number of autophagic vacuoles and in the size of the dense and residual bodies was observed in the hepatocytes of rats fasted for 24 hours; moreover, the number of dense bodies was reduced. These data suggest that the previously reported acceleration in cell protein degradation caused by fasting can be accounted for by enhanced autography. The treatment with cycloheximide, which was previously found to prevent this proteolytic response, also prevents the appearance of signs of enhanced autophagic activity.