Assessment of urine sample quality by the simultaneous measurement of urinary γ-glutamyltransferase and lactate dehydrogenase enzyme activities: possible application to unravel cheating in drugs of abuse testing.

OBJECTIVES: Evaluation of the simultaneous measurement of urinary γ-glutamyltransferase (γGT) and lactate dehydrogenase (LDH) to discriminate fresh from previously frozen specimens in urine drug monitoring. METHODS: Two widely available photometric tests (Siemens Healthineers Atellica) were used to determine the range of urinary γGT and LDH excretion and to study the decay in urinary enzyme activity under various storage conditions (room temperature, 4-8 °C, -18 °C, -80 °C). From these data, cut-off values were established and evaluated in split (fresh/frozen) specimens. RESULTS: Both assays allow robust, reliable, and simultaneous determination of urinary γGT and LDH. In healthy subjects, the 95% reference intervals for enzyme activity in native urine were γGT: 24.4-100.4 U/g Crea (creatinine) and LDH: 2.5-45.8 U/g Crea. Frozen storage for at least 7 days at -18 °C resulted in a loss of activity to less than 50% in both enzymes. Cut-offs for frozen samples were γGT≤33.2 U/g Crea and LDH≤ 8.4 U/g Crea. When applied to 100 sample pairs (fresh/frozen), 86.5% (173/200) of the measurements were conclusive and the combination of concordant enzyme measurements (low γGT/low LDH or high γGT/high LDH) was able to predict the mode of storage with a sensitivity of 96.3% and a specificity of 96.7%. CONCLUSIONS: The additional measurements of urinary γGT and LDH can be used to detect previously frozen urine specimens. A simple protocol is proposed to provide additional information on sample quality when deceit is suspected. The procedure can be easily integrated into the standard workflow of urinary drug monitoring.

Preconditioning with Maillard reaction products improves antioxidant defence leading to increased stress tolerance in cardiac cells.

Advanced glycation end products (AGEs) are considered as biomarkers of ageing and are associated with several degenerative diseases. Besides endogenous formation, significant amounts of AGEs are taken up with food. Although nutritional AGEs are considered as undesirable, proinflammatory agents, they may also enclose potentially beneficial antioxidants. We used rodent cardiac cells to evaluate if food AGEs, present in bread crust, can modify the cellular antioxidant defence. Mice were fed with bread crust containing diet to prove the in-vivo relevance for the heart. In mouse cardiac fibroblasts, bread crust extract induced a moderate elevation of ROS production causing an activation of p42/p44(MAPK), p38(MAPK) and NF-κB, followed by increased expression of antioxidative enzymes. Preconditioning studies demonstrated that this was sufficient to protect cardiac fibroblasts and rat adult cardiac myocytes against severe oxidative stress. Furthermore, mice, fed a bread crust containing diet, exhibited a similarly improved cardiac expression of antioxidative defence genes. The consumption of AGEs can therefore contribute to an improved antioxidant status of the heart, thus exhibiting cardioprotective effects in case of severe oxidative stress as in ischemia reperfusion injury. Also, these data show that the exclusive interpretation of circulating AGEs as pathophysiological biomarkers of ageing might be misleading.

Cardiac markers: a clear cause for point-of-care testing.

Point-of-care testing (POCT) in patients with ischemic heart disease is driven by the time-critical need for fast, specific, and accurate results to initiate therapy instantly. According to current guidelines, the results of the cardiac marker testing should be available to the physician within 30 min ("vein-to-brain" time) to initiate therapy within 60-90 min ("door-to-needle" time) after the patient has arrived at the emergency room or intensive care unit. This article reviews the current efforts to meet this goal (1) by implementing POCT of established biochemical markers such as cardiac troponins, creatine kinase MB, and myoglobin, in accelerated diagnosis and management workflow schemes, (2) by improving current POCT methods to obtain more accurate, more specific, and even faster tests through the integration of optical and electrochemical sensor technology, and (3) by identifying new markers for the very early and sensitive detection of myocardial ischemia and necrosis. Furthermore, the specific requirements for cardiac POCT in regard to analytical performance, comparability, and diagnostic sensitivity/specificity are discussed. For the future, the integration of new immunooptical and electrochemical chip technology might speed up diagnosis even further. However, every new development will have to meet the stringent method validation criteria set for corresponding central laboratory testing.

Maillard products as biomarkers in cancer.

Because tumors exert increased glycolysis rates, a high intracellular carbonyl stress with the formation of Maillard products may evolve. Therefore, we studied the presence of N epsilon-(carboxymethyl)lysine (CML) modification in breast cancer tissues from 20 patients and found significant cytoplasmatic staining in tumor cells that was independent of the tumor stage, tumor type, and microanatomic localization. Studying breast cancer cell lines, we also found strong cytoplasmatic CML staining that was again independent of their invasive or metastatic behavior. Our results reveal that tumor cells show a strong cytoplasmatic immunoreactivity to CML without evident association with breast carcinoma type, differentiation, tumor stage, or intratumoral localization. We conclude that CML formation is a general tumor cell-associated process.

Maillard reaction products enriched food extract reduce the expression of myofibroblast phenotype markers.

Advanced glycation end products (AGE) are associated with a wide range of degenerative diseases. The present investigation aimed at analysing the influence of AGE containing nutritional extracts on cardiac fibroblasts (CFs) as the major cell type responsible for cardiac fibrosis. Mice CFs were treated with bread crust extract (BCE) which contained significant amounts of a variety of AGE modifications. BCE treatment with up to 30 mg/mL did not impair cell viability. Furthermore, BCE induced a moderate elevation of reactive oxygen species (ROS) production and activation of redox sensitive pathways like the p42/44(MAPK), p38(MAPK) and NF-kappaB but did not alter Akt kinase phosphorylation. Expression of smooth muscle alpha-actin and tropomyosin-1, which represent markers for myofibroblast differentiation, was reduced after bread crust treatment. These data suggest a putative antifibrotic effect of melanoidin-rich food.

Air pollution-associated fly ash particles induce fibrotic mechanisms in primary fibroblasts.

Air pollution is associated with a variety of respiratory and cardiovascular disorders, including fibrosis. To understand the possible molecular mechanisms underlying this observation, we examined the effect of particulate matter on primary fibroblasts, the key regulators of the extracellular matrix. Fly ash collected in an experimental waste incinerator was used as model particles for fine and ultrafine pollution components. Brief treatment of fibroblasts isolated from adult male Wistar rat hearts with fly ash triggered the immediate formation of intracellular reactive oxygen species (ROS). Using phospho-specific antibodies we observed activation of p38 MAP kinase, p44/42 MAP kinase (ERK1/2) and p70(S6) kinase. Prolonged incubation with fly ash increased the expression of collagen 1 and TGF-beta1, but decreased mRNA levels of MMP9 and TNF-alpha. Cell proliferation was inhibited at high concentrations of fly ash. An increase in the level of advanced glycation endproduct (AGE) modification of various cellular proteins after long-term treatment of cultured fibroblasts with fly ash was observed. The results of our study demonstrate that direct activation of fibroblasts by combustion-derived particles is a mechanism that may contribute to the adverse health effects of particulate air pollution.

Role of glucoxidation and lipid oxidation in the development of atherosclerosis.

Previous data have indicated that modification of proteins/lipids by glucoxidation and/or lipid oxidation may initiate/propagate the formation of atherosclerotic plaques. Although the biomarker carboxymethyllysine (CML) has been detected in these lesions, the origin of the reactive oxygen species (ROS) leading to its formation and the source of its carbon backbone are unknown. As presented here, the stimulation of cultured monocytes by phorbol-12-myristate-13-acetate (TPA), an activator of protein kinase C that can mimic the effects of high glucose, angiotensin II, and other physiological stimuli, leads to cellular ROS generation and concomitant formation of intracellular CML. Inhibitors of ROS-generating cellular systems such as NO synthase, xanthine oxidase, or cytochrome P450 oxidase had no effect on CML formation. Likewise, in cells with inactive NAD(P)H oxidase no reduced CML formation was found. In cells exhibiting a high glycolysis rate, CML formation was unaffected. Because we found rapid CML formation in the presence of unsaturated fatty acids, it appears that lipid oxidation is quantitatively more important. In vivo studies revealed strong intracellular CML staining in areas of histiocytic/monocytic infiltration or proliferation, mostly associated with atheroma formation. Corresponding CML staining patterns were found in healing wounds of different ages, indicating that formation of atherosclerosis is a chronic wound repair associated with a low-grade inflammatory reaction. In summary, CML is formed concomitantly with oxidative stress in activated monocytes and can be regarded as a biomarker for a low-grade inflammatory tissue reaction in the atherosclerotic plaque. Its formation via lipid oxidation may be involved in the development of atherosclerosis.

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects.

We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57+/-14 years, HbA1c 8.0+/-1.8%) to 42 non-diabetic controls (C) (age 45+/-17 years). The urinary excretion of total CML in diabetic patients was increased by approximately 30% (DM: 0.58+/-0.21; C: 0.45+/-0.14 microM/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36+/-0.17; C: 0.27+/-0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).

Investigation of a capillary electrophoretic approach for direct quantification of apolipoprotein A-I in serum.

In the present study a rapid, reproducible and robust capillary electrophoresis (CE) procedure for the quantification of apolipoprotein A-I (Apo A-I) in serum without pretreatment has been developed (total run time, 11 min). The coefficients of variation (CV; n = 10) for the relative peak area are 1.8% at a concentration of 145 mg/dL and 1.6% at 196 mg/dL; and for the inter-assay 8.9% at 161 mg/dL (10 consecutive days), i.e., similar to the CVs of a high-throughput immunonephelometric routine assay. The CV for the migration time is 0.4% (n = 20). The robustness of the CE approach was tested in patient samples with hemolysis, hyperbilirubinemia and hyperlipidemia. A comparison of 99 Apo A-I serum values with results of a fixed-time immunonephelometric routine assay showed a positive constant bias of 60% (mean) for the immunonephelometric values, no deviation from linearity, but significant deviations in several samples. Investigations on interferences in the CE analyses gave no evidence that CE failed. Our study shows that CE is amenable to a fast analysis and a reproducible and reliable quantification of Apo A-I level in sera of various clinical samples.

Evidence for a novel TGF-beta1-independent mechanism of fibronectin production in mesangial cells overexpressing glucose transporters.

Recent experimental work indicates that the hyperglycemia-induced increase in mesangial matrix production, which is a hallmark in the development of diabetic nephropathy, is mediated by increased expression of GLUT1. Mesangial cells stably transfected with human GLUT1 mimic the effect of hyperglycemia on the production of the extracellular matrix proteins, particularly fibronectin, when cultured under normoglycemic conditions. Our investigation of the molecular mechanism of this effect has revealed that the enhanced fibronectin production was not mediated by the prosclerotic cytokine transforming growth factor (TGF)-beta1. We found markedly increased nuclear content in Jun proteins, leading to enhanced DNA-binding activity of activating protein 1 (AP-1). AP-1 inhibition reduced fibronectin production in a dosage-dependent manner. Moreover, inhibition of classic protein kinase C (PKC) isoforms prevented both the activation of AP-1 and the enhanced fibronectin production. In contrast to mesangial cells exposed to high glucose, no activation of the hexosamine biosynthetic, p38, or extracellular signal-related kinase 1 and 2 mitogen-activated protein kinase pathways nor any increase in TGF-beta1 synthesis could be detected, which could be explained by the absence of oxidative stress in cells transfected with the human GLUT1 gene. Our data indicate that increased glucose uptake and metabolism induce PKC-dependent AP-1 activation that is sufficient for enhanced fibronectin production, but not for increased TGF-beta1 expression.

Immunohistochemical demonstration of -(carboxymethyl)lysine protein adducts in normal and osteoarthritic cartilage.

N(epsilon)-(carboxymethyl)lysine (CML) is an advanced glycation end product formed by non-enzymatic glycation and oxidation of proteins. The distribution pattern of CML-modified proteins in normal and osteoarthritic (OA) cartilage was investigated using specific antibodies. In healthy articular cartilage, immunoreactivity for CML was preferably found in the extracellular matrix (ECM) of the superficial layer. In OA samples, CML immunoreactivity was not restricted to the ECM of the superficial layer. Interestingly, OA chondrocytes showed a remarkable cytoplasmic immunoreactivity for CML. With the help of a western blot analysis CML-modified proteins between 68 and 39 kDa could be demonstrated in OA cartilage samples. These results suggest that the accumulation of CML adducts contributes to the matrix damage in osteoarthritis. Therefore, the inhibition of CML accumulation may represent an effective therapeutic strategy to prevent severe OA cartilage injury.