Optimization of a time-resolved immunofluorometric assay for tumor-associated trypsin inhibitor (TATI) using the streptavidin-biotin system

We have developed two 'sandwich'-type time-resolved immunofluorometric assays (IFMA) for tumor-associated trypsin inhibitor (TATI) using monoclonal and polyclonal antibodies. In the standard assay the monoclonal antibody was immobilized onto the walls of polystyrene microstrip wells and the polyclonal reagent was labeled with a europium chelate. We tested various assay conditions in order to optimize the assay for sensitivity and measuring range. Purification of the labeled antibody by hydrophobic interaction chromatography was found to be the most important single factor affecting sensitivity. Assay sensitivity and range were also improved by acid treatment of the solid phase antibody. To improve the sensitivity further the streptavidin/biotin (SAB) system was incorporated into the IFMA technique. In this simple and fast streptavidin/biotin IFMA (SAB-IFMA) we used streptavidin-coated wells to which we added biotinylated monoclonal antibody and a serum or urine sample. After incubation for 1.5 h and washing, the polyclonal europium-labeled tracer antibody was added. After incubation for 1 h the wells were washed and the Eu fluorescence measured. The assay performance of the SAB-IFMA was compared to the standard IFMA and radioimmunoassay (RIA). The detection limit was 0.05 microgram/l and the analytical range 3000-fold. The mean analytical recovery was 101%. Other advantages of the SAB-IFMA were high sensitivity and the low amounts of monoclonal antibody required, only 1/50 of that used in the standard IFMA.     

Distribution of serum lipoprotein(a) levels--a non-parametric analysis

Persisting postoperative pain of the thigh is a common problem of cementless hip endoprostheses occurring in about 15-20% of the patients. We performed a comparative study including patients with (n = 40) and without (n = 45) pain of the thigh. 85 cementless porous-coated anatomic (PCA) hip endoprostheses in 74 patients were examined. All patients underwent clinical examination including a standardized questionnaire, x-ray, and 3-phase bone scintigraphy. Slight or moderate 99mTc-MDP uptake in the area of the greater and lesser trochanter as well as at the tip was a common finding in PCA prostheses in patients without pain and was not a sign of loosening of the hip. Radiologically, there was no difference between patients with and without pain. However, persisting pain of the thigh in patients with PCA prosthesis corresponded with an increased uptake at the tip and the medial and lateral femur, not being a sign of loosening even in this group. The special biomechanical conditions of cementless prostheses causing inhomogeneous intraosseous stress distribution are supposed to be the reason for that.     

Competitive time-resolved immunofluorometric assay for quantifying carbonic anhydrase VI in saliva

A competitive time-resolved immunofluorometric assay sensitive and robust enough for quantifying human salivary carbonic anhydrase isoenzyme VI (HCA VI) was developed. The solid-phase immunoassay is based on competition between Eu(3+)-labeled HCA VI and salivary HCA VI for polyclonal rabbit anti-HCA VI antibodies that are attached to microtiter plate wells precoated with sheep anti-rabbit IgG. The subsequent immunoassay including the separation of free and bound HCA VI requires only one incubation step, after which the Eu3+ of the bound labeled antigen is released into an enhancement solution. The highly fluorescent Eu chelates formed in this solution are then quantified by time-resolved fluorometry (Delfia). The time-resolution principle effectively obviates possible interferences from complex biological material such as saliva. The assay detection limit was 1.5 micrograms/L. Intra- and interassay imprecisions (CVs) were 5.1% and 5.3%, respectively. The mean analytical recovery was 93%. The mean +/- SD concentration of HCA VI in paraffin-stimulated saliva was 6.8 +/- 4.3 mg/L (n = 30) and the secretion rate was 10.2 +/- 7.9 micrograms/min. The method was useful for further investigations of the role of HCA VI in difficult matrices, e.g., saliva.     

Effects of isotretinoin therapy on lipoprotein (a) serum levels

BACKGROUND: Increases in plasma concentrations of lipids, triglycerides, and liver enzymes have been reported in patients on isotretinoin therapy. Lipoprotein (a). (Lp (a)), a cholesterol-rich plasma lipoprotein, influences the clotting system and is related to premature coronary heart disease and stroke. METHODS: Blood (7 mL) was obtained from 30 patients with cystic acne before and 30 days after the initiation of oral isotretinoin (0.5 mg/kg/day). RESULTS: An increase in liver enzymes and lipids, except high density lipoprotein, was found in our patients at the end of the study. The mean Lp (a) levels (initial value, 25.91 +/- 3.17 mg/dL) were statistically reduced (p < 0.0001) at the end of treatment (14.80 +/- 2.35 mg/dL). CONCLUSIONS: It is suggested that isotretinoin could be used as an Lp (a) lowering agent in the future.     

Increased serum lipoprotein(a) levels in patients with early renal failure

Different dermatophytes occurring primarily in animals may be transmissible to man and produce human disease; such zoophilic fungi should be considered a possible cause of skin lesions of unclear origin. Several species including Trichophyton verrucosum, Trichophyton mentagrophytes and Microsporum canis may infect human skin, causing a variety of signs and symptoms. People who have close contact to infested cattle or cats are more often exposed to fungal infections. Certain professions, such as farmers, and children are especially vulnerable. Finally, less common ways of transmission of dermatophytoses from other animal species to man are discussed.     

Apolipoprotein(a) genetic polymorphism and serum lipoprotein(a) concentration in patients with peripheral vascular disease

Serum lipoprotein(a) (Lp(a)) levels were measured in 89 men with peripheral vascular disease (PVD) and 129 (100 male and 29 woman) healthy controls. Apolipoprotein(a) genetic polymorphism was determined by immunoblotting in all subjects. Patients with PVD had significantly higher serum Lp(a) levels than controls. Apolipoprotein(a) phenotype frequencies in patients with PVD did not differ from those of the control group. Both patients and controls with phenotype S2 had higher serum Lp(a) levels than those with phenotype S4. It should be emphasized that serum Lp(a) levels were significantly higher in PVD patients than controls for those with phenotype S2, S3/S4 and S4. Raised serum Lp(a) levels together with other lipoprotein abnormalities in patients with PVD imply a high cardiovascular risk. Genetic polymorphism clearly influences serum Lp(a) levels both in patients and controls. In patients with PVD, environmental and/or other genetic factors must play a role in raising Lp(a) levels.     

Elevated levels of serum lipoprotein(a) and apolipoprotein(a) phenotype are not related to pre-eclampsia

BACKGROUND: Pre-eclampsia might result from a less effective invasion of trophoblast cells in the myometrium, caused by attenuated immunosuppression in the spiral arteries, resulting from inhibition of plasmin-mediated activation of transforming growth factor-beta-like substances. In vitro evidence indicates that lipoprotein(a) is capable of inhibiting plasmin-mediated activation of transforming growth factor-beta. Thus, high plasma levels of lipoprotein(a) might result in increased incidence of preeclampsia. METHODS: The patient group consisted of 39 patients with a history of pre-eclampsia in a previous pregnancy: Forty-seven women without pre-eclampsia in their history and matched for age were the control group. All participants gave their informed consent. In both the patient and control group blood pressure, CRP, urinalysis, cholesterol, HDL-cholesterol, triglycerides, lipoprotein(a) level and apolipoprotein(a) phenotype were determined. RESULTS: None of the participants had elevated CRP levels, excluding acute phase related elevations of lipoprotein(a). Proteinuria was present in 33% of patients and in 11% of controls (p=0.01). However, no relation was observed between proteinuria and Lp(a) level. Median Lipoprotein(a) levels in both groups were equal (300 mg/l vs. 275 mg/l; p=0.48), as well as the apo(a) phenotype distribution in both groups. CONCLUSIONS: Lipoprotein(a) and apolipoprotein(a) phenotype do not contribute significantly to the pathogenesis of pre-eclampsia.     

Elevated serum lipoprotein (a) levels associated with ulcerative colitis in a young Japanese patient

Thromboembolism has been shown to play a role in the pathogenesis of inflammatory bowel disease (IBD). A possibility exists that lipoprotein (a) [Lp(a)], a newly-discovered prothrombotic factor, also participates in the development of at least some cases of IBD. Marked elevation of serum Lp(a) levels was observed in a young patient with ulcerative colitis. A biopsy specimen of the rectal mucosa showed findings compatible with ulcerative colitis, as well as small vessel thrombus occurring within the muscularis mucosa in the rectum. Serum Lp(a) levels were markedly elevated on admission (71 mg/dl), with a gradual decrease to 46 mg/dl on discharge. Moreover, serum Lp(a) levels decreased in parallel with clinical improvement. In the quiescent clinical stage, no small vessel thrombus was observed in the mucosa on follow-up colonoscopy. The association between IBD and hyper-Lp(a)-emia would be presumable but it has been, to our knowledge, previously unreported. The case reported here would be the first young patient, suggesting the presence of hyper-Lp(a)-emia and small vessel thrombus formation occurring in association with the development of ulcerative colitis.     

Homogeneous assay for measuring low-density lipoprotein cholesterol in serum with triblock copolymer and alpha-cyclodextrin sulfate

We have developed a fully automated method for measuring LDL-cholesterol (LDL-C) in human serum without the need for prior separation, using a nonionic surfactant, polyoxyethylene-polyoxypropylene block copolyether (POE-POP), and a sodium salt of sulfated cyclic maltohexaose, alpha-cyclodextrin sulfate. Of the surfactants tested, POE-POP with a higher molecular mass of the POP block and a greater hydrophobicity reduced the reactivity of cholesterol in lipoprotein fractions; the reactivity in descending order was LDL > VLDL > chylomicron approximately HDL. Gel filtration chromatographic studies revealed that POE-POP removed lipids selectively from the LDL fraction and allowed them to participate in the cholesterol esterase-cholesterol oxidase coupling reaction system. By contrast, alpha-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and VLDL. A combination of POE-POP with alpha-cyclodextrin sulfate provided the required selectivity for the determination of LDL-C in serum in the presence of magnesium ions and a small amount of dextran sulfate without precipitating lipoprotein aggregates. There was a good correlation between the results of LDL-C assayed by the proposed method and the beta-quantification reference method involving 161 sera with triglyceride concentrations ranging from 0.3 to 22.6 mmol/L.     

Apolipoprotein(a) binds to low-density lipoprotein at two distant sites in lipoprotein(a)

Lipoprotein(a) [Lp(a)] consists of low-density lipoprotein (LDL) and apolipoprotein(a) [apo(a)] linked with a disulfide bond. Scanning force microscopy (SFM) of Lp(a) showed, for the first time, a belt-like structure of apo(a) with both ends attached to a spherical LDL. The two ends of apo(a) were bound to the LDL sphere at two distant sites. Occasionally, the ends were attached to two touching spheres. Under the same imaging conditions, LDL appeared as individual spheres. Electron microscopy (EM) studies of Lp(a) by several groups over the past decade failed to reveal this belt-like structure of apo(a). Images of isolated apo(a) in air or in phosphate buffer showed apo(a) as individual belts, and these belts tended to crowd together. Lp(a) formed leaf-like aggregates; apo(a) aggregates were fishnet-like, whereas LDL aggregates were less characteristic. Quantitative analysis of Lp(a) showed the diameter of the LDL to be 24.8 +/- 8.7 nm (n = 46), which is close to the reported value of 24.2 +/- 4.2 nm found with EM. The length of the belts attached to the spheres was measured to be 173.5 +/- 6.6 nm (n = 15). I also found, by using a functionalized tip, that the interaction force between apo(a) and its ligand, lysine, was related to the ionic strength of the bulk solution. This force can be reduced by the presence of epsilon-aminocaproic acid.     

Time-resolved fluoroimmunoassay for the determination of lisinopril and enalaprilat in human serum

A solid-phase immunoassay with detection based on time-resolved fluorescence (TR-FIA) has been developed for the determination of lisinopril and enalaprilat in human serum. The immunogen was prepared by coupling lisinopril to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to both lisinopril and enalaprilat was used. The assay is based on the competitive immunoassay principle in which the drug competes with biotin-labeled drug for a limited quantity of primary antibody bound via sheep anti-rabbit globulin to the wells of microtitration strips. At the end of the first incubation, the unbound biotin-labeled drug is washed away. In the second step, europium-labeled streptavidin (specific to biotin) reacts with the biotin already bound to the solid-phase antibody. After a washing step, the addition of an enhancement solution dissociates the europium ions from the labeled streptavidin into solution. The fluorescence from each sample is inversely proportional to the concentration of the drug in the sample. The assay demonstrates good accuracy, reproducibility and specificity at serum concentrations down to 0.5 ng ml-1. However, the useful concentration range of TR-FIA is much narrower than that obtained by double antibody radioimmunoassay (RIA).     

Effect of sample storage on quantitation of lipoprotein(a) by an enzyme-linked immunosorbent assay

This study evaluated the effect of storage on the quantitation of lipoprotein (Lp)(a) in 25 serum samples. Aliquots of serum were stored for up to three years at either -20 degrees C or -70 degrees C and Lp(a) subsequently analyzed using an enzyme-linked immunosorbent assay kit. Concentrations of Lp(a) declined during storage, and the temperatures employed elicited significantly different (P < 0.05) values within 12 mon which further diverged during three years of storage. Compared to baseline values, significant decreases (P < 0.05) in Lp(a) levels were evident after six months of storage at -20 degrees C with apparent losses (geometric mean) reaching 36.9% (95% confidence interval: 30.9%, 42.9%) after three years. Similarly, significantly lower (P < 0.05) Lp(a) values were recorded after six months of storage at -70 degrees C and at three years the decrease (geometric mean) was 19.1% (95% confidence interval: 14.3%, 24.0%). The losses, after three years, in terms of the arithmetic mean were 53.5 and 26.2% at -20 and -70 degrees C, respectively. Phenotype analysis suggested that large isoforms are more susceptible to degradation than smaller moieties. This may be related to the observation that apparent losses are reduced in samples containing over 8 mg/dL Lp(a). Nevertheless, Lp(a) levels in stored samples retained a strong correlation with the baseline values. These results must be considered specific for the storage conditions and analytical procedures employed.     

Variation in lipoprotein(a) concentrations among individuals with the same apolipoprotein (a) isoform is determined by the rate of lipoprotein(a) production

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein which is similar in structure to, but metabolically distinct from, LDL. Factors regulating plasma concentrations of Lp(a) are poorly understood. Apo(a), the protein that distinguishes Lp(a) from LDL, is highly polymorphic, and apo(a) size is inversely correlated with plasma Lp(a) level. Even within the same apo(a) isoform class, however, plasma Lp(a) concentrations vary widely. A series of in vivo kinetic studies were performed using purified radiolabeled Lp(a) in individuals with the same apo(a) isoform but different Lp(a) levels. In a group of seven subjects with a single S4-apo(a) isoform and Lp(a) levels ranging from 1 to 13.2 mg/dl, the fractional catabolic rate (FCR) of 131I-labeled S2-Lp(a) (mean 0.328 day-1) was not correlated with the plasma Lp(a) level (r = -0.346, P = 0.45). In two S4-apo(a) subjects with a 10-fold difference in Lp(a) level, the FCR's of 125I-labeled S4-Lp(a) were very similar in both subjects and not substantially different from the FCRs of 131I-S2-Lp(a) in the same subjects. In four subjects with a single S2-apo(a) isoform and Lp(a) levels ranging from 9.4 to 91 mg/dl, Lp(a) concentration was highly correlated with Lp(a) production rate (r = 0.993, P = 0.007), but poorly correlated with Lp(a) FCR (mean 0.304 day-1). Analysis of Lp(a) kinetic parameters in all 11 subjects revealed no significant correlation of Lp(a) level with Lp(a) FCR (r = -0.53, P = 0.09) and a strong correlation with Lp(a) production rate (r = 0.99, P < 0.0001). We conclude that the substantial variation in Lp(a) levels among individuals with the same apo(a) phenotype is caused primarily by differences in Lp(a) production rate.     

Structural basis for the pathophysiology of lipoprotein(a) in the athero-thrombotic process

Lipoprotein Lp(a) is a major and independent genetic risk factor for atherosclerosis and cardiovascular disease. The essential difference between Lp(a) and low density lipoproteins (LDL) is apolipoprotein apo(a), a glycoprotein structurally similar to plasminogen, the precursor of plasmin, the fibrinolytic enzyme. This structural homology endows Lp(a) with the capacity to bind to fibrin and to membrane proteins of endothelial cells and monocytes, and thereby to inhibit plasminogen binding and plasmin generation. The inhibition of plasmin generation and the accumulation of Lp(a) on the surface of fibrin and cell membranes favor fibrin and cholesterol deposition at sites of vascular injury. Moreover, insufficient activation of TGF-beta due to low plasmin activity may result in migration and proliferation of smooth muscle cells into the vascular intima. These mechanisms may constitute the basis of the athero-thrombogenic mode of action of Lp(a). It is currently accepted that this effect of Lp(a) is linked to its concentration in plasma. An inverse relationship between Lp(a) concentration and apo(a) isoform size, which is under genetic control, has been documented. Recently, it has been shown that inhibition of plasminogen binding to fibrin by apo(a) is also inversely associated with isoform size. Specific point mutations may also affect the lysine-binding function of apo(a). These results support the existence of functional heterogeneity in apolipoprotein(a) isoforms and suggest that the predictive value of Lp(a) as a risk factor for vascular occlusive disease would depend on the relative concentration of the isoform with the highest affinity for fibrin.     

Testosterone decreases lipoprotein(a) in men

We administered testosterone, with or without the aromatase inhibitor testolactone, to determine the effects of testosterone and its aromatization to estradiol on Lp(a) levels in normal men. Average Lp (a) values decreased by 37% during testosterone alone and by 28% when testosterone and testolactone were combined, suggesting that testosterone reduces Lp(a) in men primarily by an androgenic effect and not by its conversion to estradiol.     

High serum lipoprotein(a) levels in Korean type 2 diabetic patients with proliferative diabetic retinopathy

OBJECTIVE: To examine the possible association between serum lipoprotein(a) [Lp(a)] concentration and proliferative diabetic retinopathy (PDR) in Korean patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 412 Korean outpatients with type 2 diabetes were examined. Diabetic retinopathy was determined by an ophthalmologist using fundoscopic examination. Serum Lp(a) levels were measured by two-site sandwich enzyme-linked immunosorbent assay. RESULTS: The patients with PDR had higher serum Lp(a) levels than those with no diabetic retinopathy or with nonproliferative diabetic retinopathy (NPDR). Multiple logistic regression analysis showed that high serum Lp(a) levels and the presence of diabetic nephropathy were independent variables having a statistically significant association with PDR. CONCLUSIONS: Korean type 2 diabetic patients with PDR had higher serum Lp(a) levels versus those with no diabetic retinopathy or with NPDR. Although these results suggest that Lp(a) might play a role in the occlusion of retinal capillaries leading to PDR, further prospective studies are required to prove the causal relationship.