Influence of renal biomarker variability on the design and interpretation of occupational or environmental studies.

OBJECTIVES: To quantify and identify sources of within- and between-subject variability of microalbumin, N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP), three biomarkers used for early detection of renal injury, and to assess the consequences of this variability for the design and power of epidemiological studies. METHODS: Urinary excretion of microalbumin, NAG, AAP and creatinine as well as blood pressure (BP) were measured three times over a 2-year period among 142 healthy male workers. To minimise physiopathological and analytical sources of variation, standardised methods were used for urine collection and assays, and severe exclusion criteria were applied. At the first and third examinations, subjects completed the same questionnaire, providing information about their personal characteristics, tobacco and alcohol consumption, and health. A linear mixed model was used to estimate the within- and between-subject variance components and to analyse the relation between subjects' characteristics and the biomarkers. RESULTS: No change in the mean value of any of the biomarkers was observed over the 2-year period. Intra-class correlation coefficients between repeated measurements were 0.53, 0.57 and 0.56 for microalbumin, NAG and AAP, respectively; the between-subject variance was slightly higher than the within-subject variance. Subjects' age, BP, body mass index and smoking and drinking habits explained 7.2%, 12.5% and 4.2% of the total variance of microalbumin, NAG and AAP, respectively. CONCLUSIONS: In this healthy population of male workers, day-to-day differences in biomarker values appeared to be nearly as great as differences between subjects. The within-subject variance of these biomarkers is not high enough to justify systematic repeated measurements in epidemiological surveys. But, in some situations where the number of subjects is limited, measuring the subjects twice may improve study power by reducing the total variance by about 25% for each biomarker. Taking the above covariates into account would slightly improve study power and the accuracy of parameter estimates for NAG, but would add little to the analysis of microalbumin and AAP.

Contrasting renal effects of nicotine in smokers and non-smokers.

BACKGROUND: Cigarette smoking is associated with acute increase in arterial pressure due to systemic vasoconstriction and decreased skin and coronary blood flow. Virtually all cardiovascular effects of cigarette smoking are due to nicotine. However, whether nicotine also affects the renal circulation and function in humans is at present unknown. METHODS: In the current study the acute effects of a 4-mg nicotine gum on arterial pressure, heart rate as well as renal haemodynamics and function were assessed in non-smokers and chronic smokers. RESULTS: In non-smokers, mean arterial pressure (+8 +/- 1 mmHg, P<0.001) and heart rate (+13 +/- 3 beats/min, P<0.001) increased whereas effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) decreased by 15 +/- 4% and 14 +/- 4% respectively; in addition, urinary cyclic GMP decreased by 51 +/- 12% in response to nicotine administration. In smokers, mean arterial pressure and heart rate increased similarly; however, in contrast with non-smokers, ERPF and GFR remained unchanged whereas urinary cyclic GMP rose by 87 +/- 43%. Changes in ERPF induced by nicotine were positively correlated with changes in urinary cyclic GMP. CONCLUSIONS: These findings indicate that nicotine administration is associated with renal vasoconstriction in healthy non-smokers, possibly through alteration of a cyclic-GMP-dependent vasoactive mechanism. Tolerance to the renal effect of nicotine was observed in chronic smokers, despite the maintenance of the systemic response to nicotine.

The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution.

The structure of Salmonella typhimurium LT2 neuraminidase (STNA) is reported here to a resolution of 1.6 angstroms together with the structures of three complexes of STNA with different inhibitors. The first is 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (Neu5Ac2en or DANA), the second and third are phosphonate derivatives of N-acetyl-neuraminic acid (NANA) which have phosphonate groups at the C2 position equatorial (ePANA) and axial (aPANA) to the plane of the sugar ring. The complex structures are at resolutions of 1.6 angstroms, 1.6 angstroms and 1.9 angstroms, respectively. These analyses show the STNA active site to be topologically inflexible and the interactions to be dominated by the arginine triad, with the pyranose rings of the inhibitors undergoing distortion to occupy the space available. Solvent structure differs only around the third phosphonate oxygen, which attracts a potassium ion. The STNA structure is topologically identical to the previously reported influenza virus neuraminidase structures, although very different in detail; the root-mean-square (r.m.s) deviation for 210 C alpha positions considered equivalent is 2.28 angstroms (out of a total of 390 residues in influenza and 381 in STNA). The active site residues are more highly conserved, in that both the viral and bacterial structures contain an arginine triad, a hydrophobic pocket, a tyrosine and glutamic acid residue at the base of the site and a potential proton-donating aspartic acid. However, differences in binding to O4 and to the glycerol side-chain may reflect the different kinetics employed by the two enzymes.

The superiority of hollow fiber membrane over bubble oxygenator in a perfusion circuit for the evaluation of small caliber endothelialized arterial prostheses.

A perfusion circuit was constructed from a pneumatic ventricular assist device, 2 compliance chambers, 4 small-diameter silicone tubes (ID 4 mm) simulating shear inducing vascular prostheses, and an oxygenator with a heat exchanger. A bubble oxygenator (in a BO circuit) and a hollow fiber membrane oxygenator (in an MO circuit) were studied. The circuits were perfused with 30% human serum containing culture medium for 7 days at 37 degrees C. The pH, Po2, PCo2, Na+, K+, Ca2+, Cl, glucose, and total protein concentrations remained the same in BO and MO circuits during the 7 days of perfusion. The differences between the values measured in the perfusion medium and in the medium maintained in the static conditions of cell culture were not significant. In the BO circuit, the amount of cholesterol and triglyceride concentrations decreased whereas the relative amounts of albumin, alpha 1, alpha 2, beta, and gamma globulins remained stable in the perfusion medium. The medium from the BO circuit did not promote the proliferation of cultured human saphenous vein endothelial cells. In the medium from the MO circuit, the cholesterol and triglyceride concentrations did not change with perfusion time; the proliferation rate and anticoagulant function of endothelial cells were maintained. The hollow fiber membrane oxygenator preserves the biological characteristics of the cell culture medium in a perfusion circuit. The MO circuit permits the performance of relevant studies on shear stress resistance and functional activity of human endothelial cells seeded onto vascular prostheses.

A sialic acid-derived phosphonate analog inhibits different strains of influenza virus neuraminidase with different efficiencies.

A phosphonate analog of N-acetyl neuraminic acid (PANA) has been designed as a potential neuraminidase (NA) inhibitor and synthesized as both the alpha (ePANA) and beta (aPANA) anomers. Inhibition of type A (N2) and type B NA activity by ePANA was approximately a 100-fold better than by sialic acid, but inhibition of type A (N9) NA was only ten-fold better than by sialic acid. The aPANA compound was not a strong inhibitor for any of the NA strains tested. The crystal structures at 2.4 A resolution of ePANA complexed to type A (N2) NA, type A (N9) NA and type B NA and aPANA complexed to type A (N2) NA showed that neither of the PANA compounds distorted the NA active site upon binding. No significant differences in the NA-ePANA complex structures were found to explain the anomalous inhibition of N9 neuraminidase by ePANA. We put forward the hypothesis that an increase in the ePANA inhibition compared to that caused by sialic acid is due to (1) a stronger electrostatic interaction between the inhibitor phosphonyl group and the active site arginine pocket and (2) a lower distortion energy requirement for binding of ePANA.

[Early markers of nephrotoxicity: variation factors and reproducibility]. / Marqueurs précoces de néphrotoxicité: facteurs de variation et reproductibilité.

Epidemiological validity of early markers of nephrotoxicity currently used in occupational epidemiology has been poorly investigated. The aim of this study was to identify variation factors of these markers, to quantify their intra- and inter-individual variability and to evaluate the consequences of these results on study size and power. A cross-sectional survey was carried out in 1991 in a rotogravure plant including 168 male subjects (92 exposed to toluene and 76 controls). Blood and urine samples were taken twice: at study onset and one to five months later for 40% of the subjects. Creatinine and beta-2-microglobulin (beta 2M) were measured in both blood and urine; microalbumin (microALB), N-acetyl-beta-D-glucosaminidase (NAG), and alanine-aminopeptidase (AAP), in urine. Sources of physiological variation were reduced by standardization of collection and assay methods. Subjects completed a questionnaire to record information about their personal characteristics, alcohol, tobacco and drug consumption and their health. Statistical analysis of all subjects was adjusted for exposure status. Several factors were significantly related to the markers: age with beta 2M, NAG and AAP; smoking, alcohol drinking, and blood pressure with both microALB and NAG; urinary pH with beta 2M. These factors explained from 13 to 21% of the total variance of these markers. Short-term reproducibility, i.e. the correlation between the two measurements, was high for microALB (r = 0.75), moderate for NAG (r = 0.51), and low for beta 2M (r = 0.33) and AAP (r = 0.17). These results showed that confusion bias in the evaluation of exposure-marker association can be reduced by adjusting for several factors and that accuracy and study power can be improved by repeating measurements, especially for beta 2M and AAP.

Immunomodulation with low-dose immunoglobulins for moderate IgA nephropathy and Henoch-Schönlein purpura. Preliminary results of a prospective uncontrolled trial.

Recently, our group has shown that a 3-month course of intravenous immunoglobulin (2 g/kg/monthly) followed by 6 months of intramuscular immunoglobulins (IMIG, 16.5%, 0.35 ml/kg every 15 days) was able to slow or to stop the decline in the glomerular filtration rate, to reduce proteinuria, hematuria, leukocyturia and the histological index of activity on renal biopsy in patients with severe forms of IgA nephropathy (IGAN) and Henoch-Schönlein purpura (HSP). The aim of this open prospective trial was to evaluate the efficacy and safety of low-dose immunoglobulin therapy in moderate IGAN and HSP with permanent proteinuria. Fourteen patients with moderate IGAN [idiopathic IGAN: n = 11; chronic idiopathic HSP: n = 3] and permanent albuminuria were treated with polyvalent IMIG (16.5%) for 9 months (0.35 ml/kg once a week for 1 month, followed by 0.35 ml/kg every 15 days for a further 8 months). Eligibility criteria in the study were Lee histological stage I, II or III, albuminuria between 300 and 2,000 mg/day and a glomerular filtration rate > 70 ml/min/1.73 m2. IMIG were well tolerated and only 1 patient withdrew from the trial. No viral, renal or immunological side effects were observed. IMIG induced a significant decrease in albuminuria as well as in the histological activity index in the 11 cases in which a follow-up biopsy was performed. There was also a decrease in serum IgA, serum beta 2-microglobulin and IgA immune complex levels, and an increase in serum IgG1 levels. Twelve of the 13 evaluable patients improved during treatment.

Cost-effectiveness of screening for microalbuminuria using immunochemical dipstick tests or laboratory assays in diabetic patients.

To analyse the cost-effectiveness ratio of screening for microalbuminuria in diabetic patients using either dipstick tests or laboratory assays, 506 diabetic patients were screened for microalbuminuria using both a traditional laboratory assay (strategy I) or a laboratory assay only in the case of a positive dipstick result (strategy II). Dipstick pre-screening was considered positive if at least one of the tests performed by the two different operators showed an albumin excretion rate > 20 micrograms min-1. It was performed using a new dipstick, Micral-Test, designed to distinguish low albumin concentrations. Biological assay was the reference method. Costs were related to laboratory assays (strategy I) or to dipstick tests and laboratory assays for positive results (strategy II). The loss of effectiveness was related to false negative results of strategy II. The double dipstick pre-screening showed a sensitivity of 90.8% and a specificity of 80.1%. Its predictive value was 97% for a negative result and 55.6% for a positive result. False positive results were associated with elevated urinary volumes. Compared with strategy I, strategy II showed a sensitivity of 90.8%, a specificity of 100%, and predictive values of 100% and 97.5%, respectively, for positive and negative results. In a fictitious cohort of 10,000 patients, strategy II yielded a gain of 16,750 pounds on the first year, which decreased to 5345 pounds after 30 years. The loss in effectiveness was estimated at 2.38 quality adjusted life years of a diabetic patient (QALYd) initially, and decreased to 0.91 QALYd after 30 years, the annual cost-effectiveness ratio being close to 6600 pounds QALYd-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased serum and blister fluid levels of creatine kinase in patients with toxic epidermal necrolysis.

Keratinocytes have recently been reported to contain creatine kinase (CK) of brain-type isoenzyme. The aim of this study was to investigate whether necrosis of keratinocytes induced raised CK levels in toxic epidermal necrolysis (TEN). The serum and blister fluid levels of creatine kinase and its isoenzymes [muscular-type (MM), brain-type (BB), myocardial-type (MB)] were measured in 40 patients with TEN, 10 patients with other bullous dermatoses, and in suction blisters in five controls. The mean serum CK was significantly higher in TEN patients than in patients with other bullous dermatoses (mean +/- SD: 480 +/- 535 U/l vs. 107 +/- 44 U/l, P < 0.05). The MM-isoenzyme was predominant (94%). A positive correlation was found between the level of the serum CK and the percentage of body surface area (BSA) involved (r = 0.49, P < 0.001). The mean blister CK was significantly higher in TEN patients than in patients with other bullous dermatoses or controls (mean +/- SD: 728 +/- 437 U/l vs. 310 +/- 244 U/l and 268 +/- 194 U/l, respectively, P < 0.02). The isoenzyme distribution of blister CK in TEN patients was: 76.8% MM, 18.1% MB and 5% BB. Although a significant part of blister CK comigrating with CK-MB, after preincubation with protein A-Sepharose, appeared to be CK-BB/IgG complex, the CK-BB fraction constituted less than 25% of blister CK. Therefore, the CK present in increased amounts in serum and blister fluid in TEN was not directly produced by keratinocytes.

High-dose immunoglobulin therapy for severe IgA nephropathy and Henoch-Schönlein purpura.

OBJECTIVE: To determine if polyvalent IgG is promising therapy for severe IgG nephropathy. DESIGN: Open prospective cohort study. SETTING: Referral nephrology unit. PATIENTS: 11 adult patients with severe IgA nephropathy (9 who had idiopathic disease and 2 who had Henoch-Schönlein purpura) and indicators of poor prognosis. INTERVENTION: Patients were given high-dose immunoglobulins (2 g/kg each month) for 3 successive months, followed by intramuscular immunoglobulins (preparation content, 16.5%; 0.35 mL/kg every 15 days) for another 6 months. MEASUREMENTS: Histologic changes were analyzed by comparing pre- and post-therapy renal biopsy specimens blindly, using an activity index (14-point scale), a sclerosis index (10-point scale), and a semiquantitative immunofluorescence test of immune deposits. Proteinuria, hematuria, leukocyturia, enzymuria, and global renal function (creatinine and polyfructosan clearances) were evaluated before and after intervention. RESULTS: Proteinuria (median level before intervention, 5.20 g/d; median level after intervention, 2.25 g/d), hematuria, and leukocyturia decreased substantially. The decrease in glomerular filtration rate was greatly slowed or stopped (median rate of decline in glomerular filtration before, -3.78 mL/min per month; after, 0 mL/min per month). The histologic index of activity (median index before, 5; after, 2) and the staining intensity of glomerular IgA and C3 deposits also decreased. Immunoglobulin therapy was well tolerated. CONCLUSIONS: Immunoglobulin therapy may be effective in treating severe IgA nephropathy and protecting renal function. However, prospective controlled trials must confirm these preliminary results.

Proteinuria selectivity index--prognostic value in lipoid nephrosis and related diseases.

In order to predict the steroid response in lipoid nephrosis (LN), we studied age, sex, proteinuria level, histological features and proteinuria selectivity index (SI; ratio between IgG and transferrin clearances) in 52 LN cases (minimal-change disease: n = 39; focal glomerulosclerosis+IgM nephropathy: n = 13). The multivariate analysis showed that age, sex and proteinuria level were not contributive, whereas histology and SI were. The predictive value of SI was much higher than that of histological type (McFadden's r2: 47% vs. 22%, p < 0.001). Thus, SI should be systemically assessed in idiopathic nephrotic syndrome for reviewing the pathologic classification obtained by histology. However, if its prognostic value is lower than that of selectivity, initial renal biopsy remains necessary for diagnosis in adults.

[Proteinuria selectivity index: prognostic value in idiopathic nephrotic syndromes]. / L'indice de sélectivité de la protéinurie: valeur pronostique dans les syndromes néphrotiques idiopathiques.

In INS, the histological appearance constitutes a classical prognostic element: minimal-change nephropathy (MCN) responds better to treatment than focal glomerulosclerosis (FGS) or IgM nephropathy (IgMN). However, this criterion is not consistent. We evaluated the prognostic value of the proteinuria selectivity index (SI): the ratio of IgG clearance to transferrin (Tf) clearance. Proteinuria was selective for an SI less than or equal to 0.01. In the 39 MCN, the SI ranged from 0.01 to 0.39 (median 0.10) and proteinuria was selective in 21 cases. In the 13 FGS and IgMN, the SI varied from 0.05 to 0.40 (median 0.22) and proteinuria was selective in 1 case (p less than 0.01 between these two groups). The SI ranged from 0.01 to 0.17 (median 0.07) for the 25 corticosensitive (CS) forms and from 0.08 to 0.40 (median 0.20) for the 27 corticoresistant (CR) ones (p less than 0.001). Twenty-four of the 30 MCN patients and 19 of 22 cases of selective proteinuria were CS. Multivariant analysis enabled the identification of variables predictive of the response to steroids. Age, sex and level of proteinuria had no such value. The predictive value of the SI was greater than that of the histological appearance (McFadden's R-square, 47 versus 22%, p less than 0.001). When the histological aspect was known, the SI provided additional precision, but the reverse situation was not true. The predictive curve of CS as a function of the SI was sigmoidal, therefore reflecting a homogeneous distribution, despite their different histological types.(ABSTRACT TRUNCATED AT 250 WORDS)

[Proteinuria selectivity: description of a new index]. / Sélectivité de la protéinurie: description d'un nouvel indice.

An improved simple method for determination of selectivity index has been described. This new index (Creteil index) has been compared to Cameron index. The correlation between both indexes has been 0.949 (R-squared). Creteil index is a less expensive method because only two determinations are requisited in serum and urine: albumin and IgG. In Cameron index, transferrin and IgG are detected and albumin has to be measured in addition in the blood to appreciate the nephrotic syndrome.

Factors related to the electric taste threshold in type 1 diabetic patients.

To specify the factors related to taste function in Type 1 diabetes mellitus, 50 diabetic out-patients and 50 control subjects paired for age and sex were screened for taste disorders. None of them consumed significant amounts of alcohol, smoked, or had disease or took drugs capable of altering taste. Taste was studied with electrogustometry, retinopathy was detected by fluorescein angiography, nephropathy by measurement of albuminuria and microalbuminuria, peripheral neuropathy by electroneurography and electromyography, and autonomic neuropathy by cardiovascular function tests. The electrogustometric threshold was, on average, significantly higher in the diabetic group (133 +/- 30 microA) than in the control group (29 +/- 9 microA; p less than 0.001). Electric hypogeusia (electrogustometric threshold greater than 100 microA) was found among 54% of the diabetic patients vs 2% of the control subjects (p less than 0.001). In the diabetic group, the electrogustometric threshold was associated with complications of diabetes, especially with peripheral neuropathy (210 +/- 24 vs 90 +/- 22 microA; p less than 0.001) and microalbuminuria (185 +/- 25 vs 86 +/- 21 microA; p less than 0.01). It was correlated with age (r = 0.37; p less than 0.01) and duration of diabetes (r = 0.52; p less than 0.001) but not with HbA1c (r = -0.04). Using multivariate analysis, duration of diabetes and peripheral neuropathy had the strongest association with taste impairment. These results support previous findings, suggesting that taste impairment is a degenerative complication of diabetes mellitus.

[Optimization of the determination of muscle acid phosphatase in the post-mortem chicken. Study of the stability of enzyme activity during meat storage at 4 degrees C. and -20 degrees C]. / Optimisation du dosage de la phosphatase acide musculaire du poulet post-mortem. Etude de la stabilité de l'activité enzymatique lors du stockage de la viande à + 4 degr'es C et -20 degr'es C.

A colorimetric quantitative determination of total acid phosphatase in chicken muscle is proposed. Optimal conditions for the use of this enzyme were studied. This method allowed to establish that the muscular acid phosphatase of chicken does not resist to a 30 minutes incubation at 65 degrees C but that its activity is unchanged when the meat is stored at + 4 degrees C for 10 days or at -20 degrees C for 4 months.