Band 42, Heft 9

Mutations in a putative low-density lipoprotein (LDL) receptor adaptor protein called ARH have been recently described in patients with autosomal recessive hypercholesterolemia (ARH). ARH plays a tissue-specific role in determination of LDL receptor function. In the ARH gene three mismatched polymorphisms have been detected: Pro202Ser, Pro202His and Arg238Trp. These are of putative interest in plasma cholesterol level determination. To evaluate the effect of polymorphisms on plasma cholesterol levels, all polymorphisms were analyzed by PCR and restriction enzyme analysis by MnlI, HpyCH4IV and SacII in 100 Caucasian males with high (>90%, 6.29 ± 0.89 mmol/l), and 100 males with low (<10%, 3.60 ± 0.57 mmol/l), total plasma cholesterol levels. No significant differences were observed in frequencies of ARH genotypes or alleles between these two extreme groups. These results suggest that ARH polymorphisms are unlikely to be important genetic determinants of plasma cholesterol levels.

Recent evidence has denied genetic abnormality as a mechanism of the C5 variant of butyrylcholinesterase (BChE) and proposed the binding of an unknown protein with the C4 component. The present study aimed to evaluate whether the coding sequences and nontranslated sequences of the BChE gene at exons 1 to 4, 3q are structurally different in subjects having elevated BChE with and without the C5 variant phenotype. We also attempted to identify the unknown protein associated with the C5 variant and measured the BChE-specific activity in the C5 variant with an enzyme-linked immunosorbent assay (ELISA) using anti-BChE monoclonal antibody. We investigated five subjects, four of whom had elevated plasma BChE (three C5-positive [C5(+)] and one C5-negative [C5(–)]) and one control with a normal plasma BChE level. Direct DNA sequencing of the BChE gene revealed no relevant genetic mutations and no abnormal migrations in the genes of all five subjects. Precipitation of the patients’ sera with anti-human immunoglobulin A (IgA), -IgG, -IgM, anti-human albumin antibodies had no effect on the BChE activity. The measured BChE activity in C5(+) was 30 to 54% higher than the activity calculated from BChE protein content. The present results suggest that the C5(+) phenotype is not associated with any genetic abnormality in the CHE1 locus, and BChE-specific activity is enhanced in the C5(+) variant. However, the exact nature of the unknown protein related to the C5(+) phenotype remains unclear.

P-selectin and its ligand, PSGL-1, are cell adhesion molecules that facilitate interaction of platelets, leukocytes and endothelial cells. Polymorphisms of these genes have been reported to be associated with coronary heart disease (CHD). In the present study, we characterized the entire coding regions of P-selectin and PSGL-1 genes in CHD patients and healthy controls. The 17 exons of the P-selectin gene and exon 2 of the PSGL-1 gene were screened for single nucleotide polymorphisms (SNPs) by exon re-sequencing in 88 CHD patients and 96 controls. For rapid genotyping of the SNPs we developed PCR techniques with sequence-specific primers (PCR-SSP). By using PCR-SSPs we genotyped 261 CHD patients and 214 controls for 5 SNPs in P-selectin and 2 SNPs in PSGL-1 . In addition to the already described SNPs in P-selectin (S290N, N562D, V599L and T715P), we identified a novel SNP in exon 5 (V168M). The P-selectin 715P allele was more frequent among CHD patients with hypercholesterolemia compared to patients with normal cholesterol levels. A SNP (M62I) in the PSGL-1 gene was found close to the P-selectin binding site and the 62I allele revealed a higher prevalence in the control group indicating a protective effect of the mutation. The molecular characterization of P-selectin and PSGL-1 in a case-control study including CHD patients and healthy controls revealed evidence for association of the genes with development of the disease. However, the functional role of the gene variants should be elucidated by further experimental data.

Odd-numbered long-chain fatty acids (OLCFAs) increase in total erythrocyte lipids or plasma in patients with propionic acidemia and have been proposed as a useful parameter of metabolic control. However, up to now no parameter for long-term metabolic control has been available for this disorder. In contrast to previous investigations, we investigated OLCFAs with particular reference to differences for two phospholipid fractions of erythrocyte lipids. The phosphatidylethanolamine (PE) fraction of erythrocyte lipids has a slow turnover rate and might reflect the metabolic propionyl-CoA burden over a period of some weeks. Distinct increases in OLCFA of up to 200% in the phosphatidylcholine fraction were observed after episodes of metabolic decompensation and maximum OLCFA levels were found 3 weeks after the onset of a crisis. As a sign of poor metabolic control, OLCFA levels in PE steadily increased after recurrent episodes of decompensation, but not in clinically stable patients. However, in one patient with clinically good metabolic control, OLCFAs continually increased after the second year of life for an as yet unknown reason. In conclusion, data from this study indicate the usefulness of OLCFA analysis in the PE fraction of erythrocyte lipids, in particular with regard to the need for such a parameter for evidence-based improvement of management strategies in propionic acidemia.

The appearance of small, dense, low-density lipoprotein in serum has been demonstrated to be associated with increased risk of coronary artery disease. The molecular diameter of low-density lipoprotein is usually measured on the basis of mobility differences on polyacrylamide gel electrophoresis. However, since mobility assessed by this method is seriously affected by the increased levels of serum free fatty acids associated with hypertriglyceridemia, we used polyacrylamide gradient gel electrophoresis to eliminate the interference by fatty acids and devised a simple, precise method of polyacrylamide gradient gel electrophoresis to measure the diameter of small, dense, low-density lipoproteins in serum. We used apoferritin and thyroglobulin, which have a molecular diameter of 12.2 nm and 17.0 nm, respectively, and standard low-density lipoprotein particles having a diameter of 25.7 and 27.0 nm as calibrators, estimated by measurement of negative staining of electron microscopy. We also included apoferritin as an internal standard for polyacrylamide gradient gel electrophoresis. The only stain used was Coomassie brilliant blue, and it was used for lipoprotein staining. When we used low-density lipoprotein of 25.73 nm in diameter as a quality control specimen, the coefficient of variation of the size measurements obtained by our method was less than 1.2%. The new method markedly improved the laboratory procedure for measuring the diameter of low-density lipoproteins.

Lipoprotein(a) (Lp(a)) is considered an independent risk factor for atherosclerotic heart and circulatory diseases. The unique, polymorphic character of Lp(a) is based on its apolipoprotein(a) (apo(a)), which has remarkable structural analogies with plasminogen, an important protein for fibrinolysis. The formation of plasmin from plasminogen is a fundamental step in the dissolution of fibrin. Repression of this step may lead to a deceleration of fibrinolysis. It has been suggested that Lp(a) has antifibrinolytic properties through apo(a) and that the apo(a)-size polymorphism has a distinct influence on the prothrombotic properties of Lp(a). However, the results on this topic are controversial. Therefore we used a standardized in vitro fibrinolysis model to provide further information on the influence of Lp(a) on plasmin formation. Monitoring the time-course of plasmin formation, we investigated the inhibition of plasmin formation through dependence on Lp(a), respectively, free apo(a) concentration. Furthermore, we investigated the influence of three Lp(a)/apo(a) phenotypes ( 22K Lp(a), 22 kringle-4 repeats; 30K Lp(a), 30 kringle-4 repeats; 35K Lp(a), 35 kringle-4 repeats). Adding varying amounts of Lp(a) to our model, we observed that the rate of plasmin formation was inversely related to the Lp(a) concentration. At 0.1 µmol/l 30K Lp(a), for example, the plasmin formation was reduced by 12.7% and decreased further by 40.7% at 0.25 µmol/l Lp(a). A similar but more distinct effect was observed when free 30K apo(a) was added to the model (25.3% at 0.1 µmol/l vs. 59.3% at 0.25 µmol/l). Comparing the antifibrinolytic influence of different apo(a) phenotypes we found that the reduction of plasmin generation advanced with the size of apo(a). At 0.1 µmol/l Lp(a) the reduction of the plasmin formation increased in the order 22K Lp(a), 30K Lp(a) and 35K Lp(a) from 3.7% to 10.7% and 22.3%, respectively. Experiments with different phenotypes of free apo(a) showed similar results (0.5 µmol/l: 22K apo(a), 56.4% vs. 30K Lp(a), 80.4%). Summarizing these results, our study indicates a distinct interrelation of Lp(a)/apo(a) phenotype and concentration with the formation of plasmin. From the antifibrinolytic Lp(a)/apo(a) effect in vitro it may be hypothesized that Lp(a)/apo(a) also has an inhibitory influence on in vivo fibrinolysis.

Troponins are of outstanding importance for the diagnosis of myocardial infarction. Cardiac troponin T (cTnT) and the various cardiac troponin I (cTnI) assays differ with respect to method comparison, diagnostic sensitivity and diagnostic specificity. To understand the differences in the diagnostic behavior of troponin assays, AccuTnI and Elecsys Troponin STAT were used in a group of healthy men and in the follow-up of patients with aortic valve replacement (AVR). Within the healthy subjects AccuTnI was able to differentiate two subgroups from each other, whereas the cTnT concentrations of all subjects were below the detection limit. In AVR patients, cTnT and cTnI correlated sufficiently, if the postoperative periods were taken into consideration. There was a rapid increase in cTnI within 24 h. In contrast, a broad peak was evident for cTnT between 48 and 120 h. The results emphasize more the differences in the release of cTnI and cTnT from the cytoplasm and the thin filaments of the cardiomyocytes than the modifications of the troponins circulating in the blood.

We validated whether a shorter methionine loading test is as accurate as the original 6-h test in identifying hyperhomocysteinemic patients and investigated determinants of fasting and post-load homocysteine concentration. Plasma homocysteine was determined in EDTA-blood from women with a history of preeclampsia (n=106) after 12 h fasting and 3 and 6 h after an oral methionine load (0.1 g/kg body weight). The 677C > T polymorphism in the methylenetetrahydrofolate reductase ( MTHFR ) gene, vitamin B 6 , vitamin B 12 , folate and creatinine were measured as determinants of homocysteine concentration. Good correlation and agreement between 3-h and 6-h plasma concentration of post-load (r=0.93, Kendall’s tau-b=0.85) and Δ (post-load minus the fasting value; r=0.90, Kendall’s tau-b=0.79) homocysteine was observed and gross misclassification did not occur after division of 3-h and 6-h homocysteine scores into quartiles. Multiple linear regression revealed MTHFR 677 TT (p=0.01), folate (p=0.04) and vitamin B 12 (p=0.06) as determinants of fasting homocysteine concentration; only MTHFR 677TT was related to 3-h (p=0.04) and 6-h (p=0.004) post-load homocysteine concentration. The MTHFR 677TT genotype resulted in > 30% higher fasting and 3-h and 6-h post-load homocysteine concentrations compared to the wildtype CC genotype. This study shows that the 3-h methionine loading test is as good as the 6-h methionine loading test in identifying hyperhomocysteinemic patients. Furthermore, remethylation parameters ( MTHFR 677C > T) strongly affect both fasting and post-load homocysteine.

The association between elevated plasma levels of homocysteine (Hcy) and nutritional status has been shown in Alzheimer disease (AD) patients and also in vascular dementia (VaD). Moreover, a previous study provided evidence that the relation between a high Hcy level and low vitamin B 12 and folate levels in AD patients is due to biochemical damage, rather than a nutritional deficit. The purpose of this study was to investigate the relationship between plasma Hcy levels and vitamins involved in its metabolism in AD and VaD. Twenty-two VaD patients, 22 AD patients and 24 healthy subjects were studied for Hcy, vitamin B 12 , vitamin B 6 and folate. All patients and control subjects were comparable for age, educational level, nutritional and socioeconomic status. None of them showed macrocytic anemia or impaired renal function. Hcy was significantly increased in VaD patients (26.0±6.58 µmol/l) as compared to controls (10.7±3.0 µmol/l) and AD patients (22.3±4.51 µmol/l; p<0.001); however, AD patients also showed increased levels of Hcy. Folates were significantly reduced in both VaD (10.8±2.81 nmol/l) and AD (10.0±2.72 nmol/l; p<0.001) patients, while vitamin B 12 showed significantly reduced levels only in AD patients (392.1±65.32 pmol/l; p=0.02). Vitamin B 6 was not significantly different in the three groups. Increased levels of Hcy associated with low vitamin B 12 plasma levels were found only in AD patients. This observation led us to consider that vitamin B 12 metabolism does not represent the direct consequence of the nutritional status and suggests that neuronal damage results in a functional vitamin B 12 deficiency, as emphasized by recent reports. New therapeutic strategies are necessary, considering that available pharmaceutical forms of vitamin B 12 are not utilized by neurons in oxidative stress conditions.

Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance during pregnancy. In Denmark the health service offers selective screening for GDM, i.e., admission to an oral glucose tolerance test (OGTT) after pre-screening with interview for clinical risk factors for GDM, two capillary fasting blood glucose (cFBG) measurements and a urine test for glucosuria. The aim of the present study was to investigate the power of the pre-screening to identify GDM and the screening to predict adverse clinical outcome. A retrospective investigation of pregnant women undergoing screening during 1998 at Vejle County Hospital, Denmark was undertaken. The two most frequent pre-screening criteria for OGTT were body-mass index (BMI) ≥ 27 kg/m 2 and age ≥ 35 years. The highest odds ratio (OR) of 9.07 (95% CI: 2.60 to 63.70) for GDM had glucosuria and the lowest (zero) had cFBG. The frequency of complicated delivery was similar in GDM (58%) compared to non-GDM (56%). The best predictor of complicated delivery was a BMI with OR=1.50 (95% CI: 0.87 to 2.60) for BMI ≥ 27 kg/m 2 vs. < 27 kg/m 2 . The best predictor of adverse neonatal outcome was a capillary blood glucose 120 min after glucose load (cBG 120 min ) ≥9.0 mmol/l (OR=3.18, 95% CI: 1.14 to 8.89). The intermediary endpoint GDM was not superior for predicting adverse maternal and neonatal outcome. The cumulative probability distribution of cBG 120 min after a 75 g glucose load was not homogeneously distributed in groups stratified according to maternal and foetal outcome. A changed slope was seen after cBG 120 min 9.0 mmol/l. Screening cFBG of 4.1 mmol/l was unable to predict GDM and adverse outcome. Glucosuria was too rare to be effective as a screening tool. Pre-screening did not identify GDM. The best predictor of complicated delivery was a high BMI. The best predictor of foetal adverse outcome was cBG 120 min ≥ 9.0 mmol/l after a 75 g glucose load. Identical fraction complications were present in GDM and non-GDM. A refinement of the screening procedure is highly needed, and this has been initiated in Denmark.

Measurement of reproductive hormones in urine is a practical way of obtaining large amounts of information; however, there is still controversy on how to overcome problems derived from volume fluctuations between samples. Creatinine adjustment is a widely accepted solution, however, it introduces an extra cost, and large studies involving multiple sequential determinations would benefit from more economical solutions. We determined the value of creatinine adjustment, and compared it with a mathematical method that uses the smoothed profile of pregnanediol (PdG) as a reference to adjust other hormonal markers. To do this, we investigated the effects on three major urinary reproductive hormonal markers (luteinizing hormone (LH), estrone 3-glucuronide (E1G) and PdG) in 17 complete menstrual cycles. Detection of the day of LH peak did not differ between raw and adjusted data. Creatinine adjustment reduced variation in pre-ovulatory E1G levels between individuals, though the effect was negligible within individuals. No significant differences were found regarding post-ovulatory PdG rise. Although creatinine adjustment significantly reduces variability, producing smoother profiles, an equivalent degree of smoothness is obtained using the PdG adjustment. We conclude that under the current technology, for the retrospective study of urinary hormonal profiles in the human menstrual cycle, PdG adjustment is a valid alternative to creatinine.

The aim of this study was to compare the diagnostic utility of complexed prostate-specific antigen (cPSA) with total PSA (tPSA) in screening for prostate cancer. Serum concentrations of tPSA and cPSA were measured in 4479 adult men during the prostate cancer screening program in the Saarland region (Germany). The percentage of men with c/tPSA ratio above the cut-off value of 0.75 increased with increasing tPSA intervals: tPSA 0–0.9 µg/l, 4.4%; 1.0–1.9 µg/l, 24.3%; 2.0–2.9 µg/l, 43.9%; 3.0–3.9 µg/l, 50.4%; and 4.0–20 µg/l, 60.2%. The commonly accepted tPSA cut-off value of 3.9 µg/l matched to the 93rd percentile of the overall population (corresponding cPSA value, 2.9 µg/l). A total of 202 men out of 313 with increased cPSA had increased c/tPSA ratio (cut-off ≥ 0.75) vs. 186 out of 312 men with increased tPSA. Thus, an additional 16 men at high risk for prostate cancer were selected only if cPSA was utilised as a first line parameter. Our data show that, compared to tPSA, cPSA measurement will always detect more high-risk patients, independent of the cut-off levels utilised for cPSA, tPSA and c/tPSA ratio. cPSA is more effective than tPSA in selecting subjects with an elevated c/tPSA ratio who are at high risk of prostate cancer. Thus, cPSA might be seen as the superior first-line parameter in screening for prostate cancer. Using lower cut-off values for tPSA or cPSA than the commonly accepted values seems reasonable for screening purposes.

Dynamic exercise strongly affects atrial natriuretic peptides (ANP), in particular the mature bioactive αANP and the proANP fragments, namely proANP 1–98 , proANP 1–30 and proANP 31–67 . The proANPs influence kidney functions and their plasma levels increase after physical exercise. We measured urinary proANP 1–30 and proANP 31–67 levels before and at the end of physical exercise in 28 well-trained male cyclists. For the first time, the proANP 1–30 and proANP 31–67 urinary levels in athletes before and at the end of a prolonged agonistic bicycle race were measured. Urinary creatinine and total proteins were also measured. The urinary proANP 31–67 , creatinine and total protein levels were significantly higher at the end of exercise than before. In contrast, proANP 1–30 /protein and proANP 31–67 /protein ratios decreased after exercise. Even proANP 1–30 /creatinine and proANP 31–67 /creatinine ratios were lower after exercise. A significant correlation between proANP 1–30 and proANP 31–67 urinary levels at the end of exercise was found. The proANP 31–67 /creatinine ratio before and after exercise also showed a significant correlation. The variation of urinary proANP fragments confirmed their possible role in physical exercise. In particular, it could be interpreted as a response of the body or kidney to renal impairment occurring during exercise.

Increased use of designer drugs (amphetamines and amphetamine-like substances) raises the need for fast screening tests in urine in clinical settings, workplace and drug rehabilitation. Immunological assays currently used are subject to unwanted crossreactivities, partly depending on the cutoff concentrations used. The values recommended in Europe and the USA are 500 and 1000 ng/ml, respectively. In Switzerland, the recommended concentration of 300 ng/ml results in a high rate of false-positive urine samples and expensive, time-consuming confirmation testing. Using the Abbott Axsym analyzer, we found numerous false positives from patients in rehabilitation centers due to concomitant medication. Therefore, the diagnostic sensitivity and specificity of the Abbott test at different cutoff concentrations and the sensitivity of the Roche Cobas Integra, Beckman Synchron and Biosite Triage point-of-care test were examined. HPLC Bio-Rad Remedi was chosen as the method of higher hierarchical order. The specificity of the Axsym analyzer (300 ng/ml) was 86%. At 500 ng/ml or 1000 ng/ml the specificity was increased to 99 or 100%, respectively, while the sensitivity only decreased from 97 to 91 or 81%, respectively. In summary, the cutoff concentration for amphetamine screening tests should not be below 500 ng/ml to avoid a high rate of false-positive results.

Lithium is the standard first line of treatment for bipolar disorder. Because of its potential toxicity, levels of the drug have to be constantly monitored. To this end, an accurate and rapid assay is thus required. Moreover, lithium measurement allows differential diagnosis in patients with hallucinations, dementia and amnesia. The lithium concentration in blood is usually evaluated by flame photometry, atomic absorption spectroscopy or ion selective electrode. The aim of this study was to evaluate the analytical performance of a new colorimetric assay for lithium. Within-day precision of a pool with a target value of 1.25 mmol/l was 2.18% (CV) and day-to-day precision was assessed using serum aliquots containing two concentrations of lithium, and CVs were 5.47 and 1.6% at 0.6 and 1.15 mmol/l, respectively. The lower detection limit (LLD) was <0.08 mmol/l. The assay seemed to be linear up to 5 mmol/l. Results of a comparison method using Deming regression were: y=1.161x–0.0075 (r=0.9879) for regression with the Flame Photometer and y=0.9729 x+0.0133 (r=0.9787) for regression with atomic absorption spectroscopy. Moreover, in this study we did not find any interference with drugs or other analytes tested. In conclusion, this assay may be a method of choice for measuring lithium blood concentrations, especially in emergency situations.