Analysis of serum homocysteine in the laboratory practice - comparison of the direct chemiluminescence immunoassay and high performance liquid chromatography coupled with fluorescent detection

Introduction: Effective diagnosis of cardiovascular diseases requires the right tools to be used enabling selective and sensitive analysis of their biomarkers. One of them is homocysteine (Hcy), nowadays determined by immunoassays and chromatographic methods. This study aims to compare the results obtained by direct chemiluminescence immunoassay (CLIA) and high performance liquid chromatography with fluorescent detection (HPLC-FD) using commercial kits. Materials and methods: Homocysteine concentration was determined in serum samples obtained from 101 individuals, using Atellica IM HCY (Siemens Healthineers, Erlangen, Germany) and HCY in plasma/serum – HPLC-FD (Chromsystems Instruments & Chemicals GmbH, Gräfelfing, Germany) tests validated for routine analysis. The latter was applied as a reference method. The comparability and agreement between the tested methods were evaluated using the Passing-Bablok (PB) regression analysis and the Bland-Altman (BA) method of the differences analysis. Results: Studies showed that CLIA gives higher Hcy concentrations (15.7 ± 4.14 μmol/L). Passing-Bablok regression analysis of the results obtained with CLIA (y) compared with HPLC-FD (x) yielded an intercept of 0.22 (95%CI: - 2.16 to 2.46) and slope of 1.58 (95%CI: 1.33 to 1.87). Bland-Altman analysis demonstrated a systematic positive bias for CLIA of 5.85 ± 2.77 μmol/L. Conclusions: Methods disagreement precludes their interchangeability. Lower Hcy values by HPLC-FD result from its greater selectivity. High performance liquid chromatography with fluorescent detection should be considered as preferential method for analysing Hcy in blood serum as well as the recommended reference method for routine clinical analysis. This fact, however, imposes the need to establish new reference ranges

Isolation and characterization of acetylated derivative of recombinant insulin lispro produced in Escherichia coli

PURPOSE: Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis. METHODS: For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed. RESULTS: Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro—one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine. CONCLUSIONS: The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro

A person with diabetes as a patient - basic rules of conduct

BACKGROUND: Diabetes is very widespread disease. More and more patients suffer from hyperglycaemia and low levels of insulin because of obesity, aging and wrong diet. The most important in treatment based on health condition is to normalize glucose level. It is recommended to treat patients to avoid many complications of hyperglycaemia, e.g. cardiovascular diseases, stroke episodes, neuropathy or nephropathy. The most common cause of death are cardiovascular problems and renal failure. MATERIAL AND METHODS: A proper review of published literature provide to define laboratory indexes, risk factors, complications to precise methods of treatment among patients with diabetes. RESULTS: The problem of growing number of people suffering from diabetes is an important issue to find solutions to conduct their treatment. Basic method is a change of diet and body loss. The most important medicament in treatment is metformin. To achieve the best results of conducting therapy should be extended by pioglitazone or liraglutide. Also addition of aspirin lower dyslipidemia problems. Such a combination provides to minimise side effects of sickness. CONCLUSIONS: Number of people suffering from DM is still and will be growing over the next years. It is significant to diagnose these people because untreated diabetes provides to many complications e.g. stroke or acute coronary syndrome. It is important to examine patients correctly what allows to turn on correct treatment. Basic standards in treatment should contain lower glucose levels, avoid complications to enhance life quality. . Expanded therapy is also concentrated on hypertension, acidosis and ketoacidosis coma to obtain beneficial effects

Changes seen in the body of an elderly patient suffering from diabetes

Background: Diabetes is a foreign disease today. This is a chronic, metabolic disease that is characterized by elevated blood glucose levels. There are many types of disease. Each of them has different characteristics. The key in the fight against the disease is to perform appropriate laboratory tests to diagnose diabetes and to introduce appropriate treatment to prevent many complications, e.g. impairment of: vision, kidneys, cardiovascular system or hearing. Material and methods: A proper review of the literature was used, which identifies risk factors, complications and proper management in diagnosing diabetes. Results: The growing problem of diabetes forces us to find the most accurate way to treat the disease. Performing reliable diagnostics is crucial. The reference method (IDMS) and methods used in routine diagnostics, oxidase hexokinase are the basic and necessary laboratory tests used in the diagnosis of diabetes. Conclusions: In the coming years, the number of patients suffering from diabetes will continue to grow. Diagnosis and treatment of this disease is extremely important, because neglecting the disease can have many serious consequences for healthy physical and mental functioning. The basis is the performance of correct tests, which allows for proper treatment

Laboratory tests in geriatric patients - an overview of the latest reports

Laboratory tests are extremely important in patient’s monitoring and/or diagnosing. We described those that are particularly significant for elderly patients, namely: peripheral blood morphology, arterial blood gasometry, cancer markers. The aim of this article is to summarize current knowledge about laboratory tests important in geriatrics, as well as draw attention to the diverse use of laboratory tests in clinical practise

A holistic approach to the problem of Rheumatoid Arthritis in geriatric patients

Rheumatoid arthritis is a chronic disease and belongs to the group of systemic connective tissue diseases having an autoimmune basis. The most commonly reported symptoms by patients are pain, swelling and difficulty moving the affected joints. The course of rheumatoid arthritis is different in the elderly, the onset of the disease in the elderly is more severe than in the young, and large proximal joints are primarily involved. Make a diagnosis as soon as possible and start treatment, the greater the chance of delaying disease progression and even remission, which will minimize disability. In the treatment of rheumatoid arthritis in the elderly, who often have co-morbidities and often mobility difficulties, the role of physiotherapist and nursing care is extremely important next to medical treatment. The use of kinesitherapy allows you to increase the range of motion of the joints and maintain them in good functional condition. Physiotherapy, on the other hand, has analgesic and anti-inflammatory effects. This effect is enhanced by properly selected pharmacological treatment, carried out with the help of disease-modifying drugs, anti-inflammatory drugs, glucocorticosteroids and biological drugs

Development, Validation, and Two-Year Application of Rapid and Simple LC-MS/MS-Based Method for the Determination of K2MK-7 in Blood Samples

Biological properties of menaquinone-7, one of the vitamin K2 vitamers (K2MK-7), both those proven and those that remain to be investigated, arouse extensive interest that goes beyond the strictly scientific framework. The most important of them is the prevention of age-related diseases, considering that we live in the times identified as the era of aging societies and many people are exposed to the vitamin K2MK-7 deficiency. Therefore, an effective analytical protocol that can be adopted as a diagnostic and preventive analytics tool is needed. Herein, a simple sample preparation method followed by the liquid chromatography-tandem mass spectrometry-based method (LC-MS/MS), was used for the selective and sensitive determination of K2MK-7 in serum samples. Under the optimized conditions, using 500 µL of serum and the same amount of n-hexane, the reproducibility and the accuracy were obtained in the ranges of 89–97% and 86–110%, respectively, and the limit of detection value was 0.01 ng/mL. This method was used for the routine analysis. Statistical interpretation of the data from 518 samples obtained during 2 years of practice allowed for obtaining information on the content and distribution of K2MK-7 in the Polish population, broken down by the sex and age groups