[Measurement of angiotensin-I-converting enzyme in the blood: help for method validation]. / Dosage de l'enzyme de conversion de l'angiotensine-I sanguine : aide à la validation de méthode.

Blood angiotensin-converting enzyme (ACE) assay is now realized by the determination of enzyme activity on synthetic substrate, mostly furylacryloyl-phenylalanyl-L-glycyl-L-glycine (FAPGG). The matrix can be serum or heparin-plasma, with or without a separator; the assay developed on serum or plasma is not adapted to other matrix such as cerebrospinal fluid where the ACE activity is much lower. This assay has been adapted on a number of automated biochemistry analyzers with the specifications of the supplier of reagents, sometimes with modification of volumes or times for analysis. Samples can be stored at +4̊C for at least for one week, freezing at -20̊C is possible but refreezing is not advised. The assay is linear from 10 to 200 UI/L. Fidelity is excellent after calibration of the assay. Accuracy can be calculated from IQA and EQA results, and the analytical uncertainty is between 2% and 5% in function of the serum ACE value. Usual values will be soon available from studies on age brackets and sex, because ACE activity seems to be more elevated in boys during adolescence. At signature, it is interesting to have medical information on the diagnosis of sarcoidosis or its treatment including ACE inhibitors as a proof of intake; we can give a commentary on elevation of serum ACE activity from other causes than sarcoidosis and the causes for low activities.

Proteinuria typing: how, why and for whom?

The typing of proteinuria is one of the complementary examinations carried out during the exploration of proteinuria. It aims to separate and identify the different proteins, or fractions of proteins, that make up proteinuria. The nature and relative importance of the proteins present reflect the location of the renal involvement and help to determine the etiology. The typing of a proteinuria also allows the detection of a monoclonal component in urine and its quantification. Finally, it allows highlighting the existence of a proteinuria of overload that can occur in the absence of kidney damage. Many methods allow the typing of proteinuria, and these have benefited in recent years from technological advances. The purpose of this review is to summarize typing methods currently used, their benefits and limitations, and the help that these diagnostic tools can provide to the management of patients.

Sweat chloride quantification using capillary electrophoresis.

BACKGROUND: Cystic fibrosis (CF) is the less rare and severe genetic disease among the European population. Biochemical diagnosis of CF is based on the demonstration of increased chloride concentration in sweat samples, obtained during the sweat test (ST). WynSep developed a capillary electrophoresis with contactless conductivity detection (CE-C4D) able to measure sweat chloride with a low sample volume. We evaluated the clinical feasibility of this device in a cohort of patients suspected of CF, in comparison with the common coulometric method (ChloroChek chloridometer). METHODS: We determined sweat chloride concentration of 65 samples from patients referred to our institution to undergo a sweat test. Each sample was submitted to coulometric method first, then WynSep-CE, with or without internal standard (IS) subject to sufficient volume sample. RESULTS: A total of 53 samples were analysed by both coulometric and WynSep-CE (using IS) methods. The method validation showed comparable analytical performances for both methods; no false positive or false negative was recorded. The two methods showed a high correlation (r = 0.993, p < 0.001) and a close agreement was demonstrated by two different statistical tests (Bland-Altman and Passing-Bablok). CONCLUSIONS: WynSep-CE is an accurate, fast, easy-to-use and an appropriate method for CF diagnosis.

Preanalytical step of urinary protein measurement: from urine sampling to preparation for analysis of the specimen.

The preanalytical phase is a key step in urinary protein measurement. It is a complex step, which includes urine sampling, storage and transport to the laboratory and preparation for analysis of the specimen. It can lead to numerous errors, since urine sampling is made by the patient himself. According to different cases, the procedures for urine sampling are presented in appendix 1 to 4. The aim of the presented guidelines is to improve the preanalytical step. In addition to the sampling made by a well-informed patient, the laboratory has to optimize transport and sample preservation, according to the analytes. The 24-hour urine collection is divided into aliquots with commercially available systems. Urinary proteins are measured on samples stored without preservatives. When 24-hour urine collection is not possible, a mid-stream urine sample is the most appropriate sample for protein measurement.

CCDC115-CDG: A new rare and misleading inherited cause of liver disease.

Congenital disorders of glycosylation (CDG) linked to defects in Golgi apparatus homeostasis constitute an increasing part of these rare inherited diseases. Among them, COG-CDG, ATP6V0A2-CDG, TMEM199-CDG and CCDC115-CDG have been shown to disturb Golgi vesicular trafficking and/or lumen pH acidification. Here, we report 3 new unrelated cases of CCDC115-CDG with emphasis on diagnosis difficulties related to strong phenotypic similarities with mitochondriopathies, Niemann-Pick disease C and Wilson Disease. Indeed, while two individuals clinically presented with early and severe liver fibrosis and cirrhosis associated with neurological symptoms, the other one "only" showed isolated and late severe liver involvement. Biological results were similar to previously described patients, including hypercholesterolemia, elevated alkaline phosphatases and defects in copper metabolism. CDG screening and glycosylation study finally led to the molecular diagnosis of CCDC115-CDG. Besides pointing to the importance of CDG screening in patients with unexplained and severe liver disease, these reports expand the clinical and molecular phenotypes of CCDC115-CDG. The hepatic involvement is particularly addressed. Furthermore, hypothesis concerning the pathogenesis of the liver disease and of major biological abnormalities are proposed.

Serum allantoin and aminothiols as biomarkers of chronic heart failure.

Background Oxidative stress (OS) represents the primary mediator of chronic heart failure (CHF) development and progression. It is well established that homocysteine is able to generate reactive oxygen species. Small amounts of allantoin in human serum result from free radical action on urate and may provide a stable marker for in vivo free radical activity. To investigate whether some easily measurable indexes such as antioxidants (uric acid, glutathione) and related molecules (allantoin, homocysteine and cysteine) can serve as OS biomarkers. Methods We investigated 75 stable CHF patients. Aminothiols and purine compound levels were determined by capillary electrophoresis. Results The homocysteine level was markedly elevated in CHF patients, whatever the aetiology. Parameters of the transsulfuration pathway and the investigated purine compounds were significantly increased. Conversely, total glutathione was decreased. The allantoin/uric acid ratio was significantly higher in CHF patients with an hyperhomocysteinaemia >17 µmol/L. All parameters of the transsulfuration and purine degadation pathways were significantly correlated, suggesting an OS in CHF patients. Conclusion Our data show an imbalance of serum aminothiols and purine compounds in these CHF patients on adapted therapy. We suggest that the evaluation and control of these new markers may help improve the OS that participates in the progression of the disease.

Homocysteine in Chronic Heart Failure.

BACKGROUND: Hyperhomocysteinemia (HHcy) is a risk factor for cardiovascular disease. Homocysteine (Hcy) can generate reactive oxygen species. Oxidative stress enhances the progression of cardiovascular diseases and has long been implicated in chronic heart failure (CHF). This study was to evaluate the predictive value of plasma Hcy levels in CHF patients and to investigate the relationship with other markers. METHODS: We investigated 134 adult CHF patients (males, 74%; mean age, 60.0 ± 14.8 years). Echocardiography, 6-min walk test, and determination of peak oxygen consumption (VO(2max)) were performed. Serum levels of Hcy and other markers were determined. Clinical follow-up was performed at five years. RESULTS: The mean Hcy level was markedly elevated in CHF patients (18.4 ± 7.83 µmol/L) vs. control subjects (12.8 ± 3.14 µmol/L; p < 0.01), whatever the etiology of heart failure (non-ischemic, n = 74, 17.6 ± 7.8 µmol/L; ischemic, n = 60, 19.3 ± 7.8 µmol/L). Hcy correlated negatively with VO(2max) and positively with BNP. Kaplan-Meier analysis showed that CHF patients with HHcy > 15 µmol/L had a significantly lower survival rate (35% vs. 56%, log-rank p < 0.05) than those without HHcy. Cox regression revealed that HHcy and hs-CRP were the most powerful independent predictors of mortality in patients at 5 years. CONCLUSIONS: HHcy is common in CHF patients and is associated with an increased risk of death at 5 years. We suggest that Hcy can be used in clinical practice as an additional risk marker in CHF patients with various medications.

[Urinary protein electrophoresis, which analysis to select? A simplified interpretation]. / Électrophorèse des protéines urinaires, quel choix analytique ? Interprétation simplifiée.

Permanent proteinuria is an early marker of the kidney dysfunction. Tracking by urinary strip, imposes a precise quantification by the laboratory. In front of the difficulties of urine collection during 24 hours, protein determination can be carried out on the urine of a miction and can be expressed as g per g of creatinine (uPCR). We analysed the impact of the expression of the proteinuria in g/L (uP) as compared to uPCR on the urinary electrophoretic profiles. A revision and a simplification of this in practice clinical interpretation is proposed. The proteinuria of 696 in-patients was quantified on an Olympus AU2700. The urinary electophoretic profiles (SDS-AGE) were interpreted by two biologists. uP and uPCR are well correlated (r=0.847, p< 0.0001). Data agreed for proteinuria > 1 g/L but concordance was obtained only in 74% of the subjects and 55% of the pathological patients. A repetition of the analyses is suggested. The interpretative diagram suggested with simplified comments improved interpretation. We advise an interpretation by two biologists. In conclusion, interpretation of the urinary electrophoretic profile rests on the rate of total proteinuria. Expression of the proteinuria as g/g of creatinine must be associated with the expression in g/L because of the analytical conditions. The SDS-AGE Technique does not allow the identification of the monoclonal compound but allows a quantitative follow-up under treatment and especially an early tracking of the type of renal dysfunction.

Simultaneous determination of allantoin, hypoxanthine, xanthine, and uric acid in serum/plasma by CE.

Allantoin (All) is an oxidative end product of purines in mammals. The small amount of All present in human plasma or serum results from free radical action on urate and may provide a stable marker of in vivo free radical activity. Because free radicals have been implicated in the development and progression of atherosclerosis, this study focused on the metabolic compounds of the All pathway. We propose a new fast CE (CE/UV) method for the simultaneous determination of All, uric acid (UA), hypoxanthine (HX), and xanthine (X) in human plasma. These products were quantified in the plasma of patients with chronic renal failure before hemodialysis (n = 6), patients with chronic heart failure (n = 6) and controls (n = 6). The filtered plasma were diluted ten-fold before the direct injection in CE/UV (195 nm), which allows separating the four compounds in less than 13 min. The metabolites were detectable at concentrations of 0.3-0.6 micromol/L. The method was linear over the range 0.5-150 micromol/L for All, HX, and X and 10-1500 micromol/L for UA (r > 0.99). The analytical performance of this method is satisfactory with intra-assay CV < 3.4%, inter-assay CV < 5% (HX and X < 7%), and recovery (93-101%). The proposed CE-UV method appears to be a useful tool for studying physiological and pathological changes of HX, UA, and All levels in plasma samples, the latter being a possible indicator of free radical damage in vivo.