Citrulline is a nonessential free amino acid, detectable in various biological fluids such as plasma, urine and cerebrospinal fluid. The plasma citrulline concentration is increasingly considered to be a reliable biomarker of enterocyte function. Current analysis usually involves lengthy HPLC separations as a part of classical amino acid profiling, or mass spectrometry usually in combination with derivatization. We employed UPLC-HILIC-tandem mass-spectrometry (MS/MS) of acetonitrile-derived supernatants from plasma samples of control subjects and of patients who had received myeloablative chemotherapy. Detection was achieved by the selected reaction monitoring of transitions: m/z 176-->70 and 180-->74 (for the deuterated standard), respectively. The method was precise and accurate with inter-day CV<3.9%(n=30), recoveries ranging from 98.0 to 100.3% and high linearity from 0.3 to at least 2000mumol/L. The results for 202 plasma samples agreed well with those obtained by the classical HPLC-fluorescence method. By a simple protein precipitation/extraction step and the UPLC separation the result can be available within 30min of receipt with a capacity of at least 12 assays per hour. Citrulline in blood and plasma or serum was stable for at least 2 days at room temperature which would permit postal transport to the laboratory. The UPLC-MS/MS method for measuring plasma citrulline concentrations is fast and robust and is therefore an ideal tool for monitoring the intestinal enterocyte capacity of patients with various pathological conditions.

Abstract Background: There is frequent discussion on the susceptibility of direct high-density lipoprotein cholesterol (HDL-C) methods to matrix effects. In Vitro Diagnostics manufacturers recognize this issue and regularly improve their HDL-C reagent formulations in subsequent generations. Methods: The 3rd generation direct HDL-C assay from Roche was investigated for matrix effects in comparison to the former generation, a Beckman direct HDL-C method and a conventional phosphotungstate (PTA)/Mg(2+) precipitation method. In addition, 235 heparin plasma samples were measured freshly and after a freeze-thaw cycle with the Roche 2nd and 3rd generation direct HDL-C. Biases, outliers, and intraclass correlation coefficients (ICCs) were calculated for both experiments. Multivariate analysis was used to investigate interference by matrix components. Results: In fresh samples, Roche 2nd and 3rd generation HDL-C methods averaged +0.15 mmol/L (95% CI: 0.13-0.16) and +0.08 mmol/L (95% CI: 0.07-0.09) higher compared to frozen samples. In frozen aliquots, ICCs for Roche 2nd and 3rd generation and Beckman direct HDL-C as compared to PTA/MgCl(2) were 0.963, 0.966, and 0.924, respectively. Predictors of outliers (defined as having an absolute difference >0.21 mmol/L) in comparisons of direct methods to the PTA/MgCl(2) precipitation method were high triglyceride and low albumin levels. Conclusions: The 3rd generation direct HDL-C from Roche has become insensitive to most matrix effects, bringing along more accurate results in hypoalbuminemic and hypertriglyceridemic samples. Surprisingly, Roche direct assays produced significantly higher HDL-C levels in fresh samples compared to frozen plasma samples. If confirmed by others, the latter finding has implications for patient management and necessitates further reagent optimization. Clin Chem Lab Med 2009;47.

The role of the apo E polymorphism in the removal of remnants of very low density lipoproteins and chylomicrons was studied after a carbohydrate-rich diet in 10 healthy normolipidemic volunteers with different apo E phenotypes during 7 days. The cholesterol concentration in the heparin-sepharose bound part of the VLDL + IDL fraction (d < 1.019 g/ml) was taken as an estimate of the remnant concentration. Before and after carbohydrate-rich diet retinyl palmitate, mixed with cream, was consumed by each subject the evening before the fasting venepuncture to quantify the removal of chylomicron remnants. After the diet there was a comparable mean rise in the three groups in serum and in very low density lipoprotein triglycerides of about 30% and 50%, respectively. The concentration of remnants of very low density lipoproteins increased slightly in all subjects. The concentration of retinyl palmitate in the d < 1.019 g/ml fraction was 20% lower than before this diet in the E-2 homozygous subjects. In the other two groups, however, 25 to 80% higher retinyl palmitate levels were found. It is concluded, that after a carbohydrate-rich diet there is only a slight increase of very low density lipoprotein remnants, independent of the apo E polymorphism. The removal of chylomicron remnants, however, seems to be facilitated in E-2 homozygous subjects, in contrast to a slower removal in the groups with other apo E phenotypes.

Proinflammatory cytokines, such as interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha), are suggested to have an important role in the process of atherosclerosis. Patients with heterozygous familial hypercholesterolemia (FH) have a marked elevation in the plasma level of low-density lipoproteins (LDL), and they show early development of atherosclerosis. The aim of the present study was to test with a whole blood culture system if hyperlipoproteinemia is associated with increased cytokine production capacity in these patients and if treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors influences this production capacity of blood cells, at both the protein and mRNA levels. The capacity of blood cells in a whole blood culture to produce IL-1beta, IL-6, TNF-alpha, IL-12, IL-18, and IL-1 receptor antagonist (IL-1Ra) in response to lipopolysaccharide (LPS) appeared to be similar for heterozygous FH patients and healthy volunteers. Furthermore, the capacity to produce IL-1beta, IL-6, and TNF-alpha in response to LPS was not modified by cholesterol synthesis inhibitors at the level of mRNA expression or at the level of release. On the other hand, the release of IL-1Ra was significantly increased after treatment with HMG-CoA reductase inhibitors, although only at the protein level. This suggests a possible beneficial anti-inflammatory role for this therapy.

Post-heparin plasma activity of lipoprotein lipase (LPL) and hepatic lipase (HL), and fat and muscle activity of LPL were measured in neutered lean and obese cats. Lipoprotein lipase, hormone-sensitive lipase (HSL), and tumor necrosis factor a (TNF) mRNA were measured in muscle and fat tissue with real-time PCR using primers for feline LPL, HSL, and TNF. Lipoprotein lipase plasma and fat activity and fat mRNA levels were significantly lower (50, 80, and 50%, respectively) in obese cats than lean cats, whereas the muscle/fat ratio of LPL was significantly higher in obese compared to lean cats. The activity of HL was not different between the groups. Hormone-sensitive lipase mRNA levels were significantly higher in obese than lean cats. The level of fat TNF also was significantly higher in obese cats than in lean cats, whereas the level in muscle was not different. The lower LPL activity and mRNA expression in fat and the higher LPL and HSL mRNA expression in muscle in obese cats compared to lean cats expectedly favor a redistribution of fatty acids from fat to muscle tissue where they can be deposited or used for energy in times of need. Tumor necrosis factor alpha may regulate this repartitioning process through suppression of adipocyte LPL.

The effect of hyperlipoproteinemia on systemic candidiasis was investigated by assessing the susceptibility of hyperlipoproteinemic, apolipoprotein E (ApoE)-deficient (ApoE -/-) mice to a systemic Candida albicans infection. The absence of ApoE in these mice resulted in an eightfold increase in plasma lipoprotein concentrations in the very low-density lipoprotein (VLDL) fraction, as compared with levels seen in ApoE +/+ mice. Mortality due to candidemia was significantly higher (86%) in ApoE -/- mice than in ApoE+/+ mice (52%), and in platings of homogenized kidney material on fungal culture medium, ApoE -/- mice yielded significantly higher levels of C. albicans outgrowth than did ApoE+/+ mice. C albicans grew twofold better in ApoE -/- plasma in 4 h than in ApoE+/+ plasma, and depletion of lipoproteins from plasma resulted in a significant seven- to tenfold increase in C. albicans growth. Recombinant ApoE did not directly inhibit C. albicans growth. Our data indicate that the increased susceptibility of ApoE -/- mice to C albicans is due both to increased growth of blastoconidia in ApoE -/- mice in response to the availability of lipids as nutrients, and to the neutralization of candidacidal factors by lipoproteins. This study suggests that lipoproteins play a significant role in host defense against candidiasis.

Physicians should be properly informed of the clinical chemistry diagnostic potential for the diagnosis and classification of hyper- and dyslipidemias by laboratory determinations of lipids, lipoproteins, and apolipoproteins. New analytes are regularly found to be relevant for screening and risk estimation for coronary artery disease in vascular medicine. These analytes can be distinguished between parameters working on the long-term or working acutely. However, in times of restricted laboratory budgets, it is not always possible to add the new analyte to the routine diagnostic supply without having answered the question of whether the new analyte indeed adds to the chronic or acute risk estimation power presently available. This is relevant for homocysteine and for C-reactive protein (CRP). Both parameters appear to be interrelated to most common cardiovascular risk factors supposed to promote atherosclerosis and to ultimately provoke cardiovascular disease, and in fact are not independent. The latter certainly has added value in acute situations. With regard to the chronic risk estimators, it must be concluded that there is a multifactorial influence, with an important contribution made by social and lifestyle factors. This review draws attention to the multifactorial aspects of coronary heart disease, risk profiling using computer programs, socioeconomic factors, and implementation problems of interventions.

Laboratory-based coronary heart disease risk assessment classically involves measurement of lipids and lipoproteins. In this review, information is provided on the methods commonly used in laboratories for the diagnosis of hyperlipidemia, including aspects of precision and accuracy. The latter, when fulfilled, allows the use of uniform reference values. Special attention is paid to the risk estimation using apolipoprotein B and lipoprotein(a) measurement. The overall aim of this review is to simplify the laboratory-based risk estimation for coronary heart disease and to provide help in interpreting the results for effective prevention and treatment of this complex disease.