Abstract: BACKGROUND: Exercise increases natriuretic peptide levels in chronic heart failure (CHF) patients, but the effect is considered minor. We assessed acute and short-term release (<24 h) of NT-proBNP in CHF patients after a maximal cardiopulmonary exercise test (CPET) and 2 different submaximal training sessions. METHODS AND RESULTS: 102 CHF patients either performed CPET (Group 1), a 1 h endurance (Group 2) or a combined endurance-resistance training session (Group 3). NT-proBNP concentration was determined before, at exercise cessation and after 18-22 h (Protocol A). In 20 patients, samples were obtained before, at exercise cessation, after 15, 30, 45, 60, 90 min, 2, 3, 4, 5, 6, 12, 22 h (Protocol B). Protocol A: At peak exercise, a 15%, 11% and 17% relative increase (p<0.001 vs baseline, all 3 groups) was seen, with a return to baseline after 18-22 h. The increase correlated with indicators of more advanced heart failure. Protocol B: A biphasic pattern was derived with a first peak within 1 h of exercise termination and a second peak (39%, 31% and 33% higher than baseline; p<0.05, all 3 groups) after 4-12 h. CONCLUSIONS: The observed biphasic release of B-type natriuretic peptides supports standardization of sampling, taking recent exercise into account.
Abstract: BACKGROUND: B-type natriuretic peptide (BNP) is used as prognostic marker in acute coronary syndromes. It is unknown whether BNP reflects the amount of jeopardised myocardium in the initial phase of acute ST-elevation myocardial infarction (STEMI). METHODS: Patients admitted for percutaneous coronary intervention in the acute phase of STEMI were studied. Samples for BNP were taken at the time of coronary intervention and were correlated with the amount of jeopardised myocardium. This was defined on coronary angiography as the vascular region distal to the infarct-related lesion and was expressed by a vascular score. The extend of epicardial injury on ECG was evaluated by: summation of ST-segment elevation in the infarct-related leads, number of leads with > or = 1 mm ST-segment elevation and number of leads with > or = 1 mm ST-segment depression/elevation. RESULTS: A total of 113 patients (median age (25th, 75th percentile) 61 (54, 69)) were studied. Median BNP was 27 pg/ml (12, 62) and was assessed 225 min (150, 315) after onset of pain. There was no significant relation between BNP and vascular score (r=0.026, p=0.8). The only independent variable of BNP was time delay between onset of pain and sample collection. Stepwise regression identified the extent of ST-segment elevation as independent predictor of the vascular score. CONCLUSION: At initial presentation of STEMI, BNP measurement does not allow a correct prediction of jeopardised myocardium. In contrast, ST-segment analysis, in particularly the extent of ST-segment elevation, provides useful information about the extent of jeopardised myocardium.
Abstract: The aim of this study was to test the influence of high-dose folic acid (10 mg/d) on endothelial function in patients referred for coronary intervention after an acute myocardial infarction (AMI) and determine its relation to homocysteine levels. Flow-mediated dilation (FMD) of the brachial artery was performed in 40 patients after AMI (16 with normal homocysteine levels and 24 patients with elevated levels [>11 micromol/L]). Subjects were randomized to receive first folic acid (10 mg/day; group A) or placebo (group B) for 6 weeks in a double-blind crossover trial with a 2-week washout. Plasma folate, total homocysteine and its subtypes (oxidized, reduced, and protein-bound), FMD, and nitroglycerin-mediated dilation were assessed at baseline and at 6 and 14 weeks. In group A, folic acid improved FMD from 3.98 +/- 0.35% to 6.44 +/- 0.56% (p <0.001). This effect persisted after the crossover with placebo (5.42 +/- 0.59, p = 0.13). In group B, placebo did not increase FMD (4.01 +/- 0.34% vs 4.46 +/- 0.38, p = 0.38); however, a significant increase was observed in the second active treatment period (6.49 +/- 0.56%, p = 0.005). In both groups, improved FMD neither correlated with basal levels of homocysteine and its subtypes nor with changes induced during the folate treatment. Nitroglycerin-mediated dilation did not change significantly in either group. Folic acid increased FMD in both normo- and hyperhomocysteinanemic groups (p = 0.006 and p <0.001). In conclusion, 6-week treatment with high-dose folic acid improves endothelial function in post-AMI patients, independent from homocysteine status. Folic acid can be recommended to improve postinfarction endothelial dysfunction in patients with normo- and hyperhomocysteinemia.
Abstract: The remnant kidney rat model has been extensively used for the evaluation of bone changes due to uremia. The present study aimed to assess the effect of the dietary phosphorus availability and of the severity of renal failure on bone histomorphometric changes and various biochemical markers over time in this model. Chronic renal failure (CRF) was induced in male Wistar rats by 5/6th nephrectomy. Half of the number of animals received a standard rat diet (STD) (0.67% P, containing low bioavailable phosphorus of plant origin); the other animals were fed a high phosphorus diet (HPD) (0.93% P, containing inorganic phosphorus with high bioavailability). Every two weeks, blood and urine samples were collected. At sacrifice after 6 or 12 weeks, bone samples were taken for the measurement of histological and histodynamic parameters. Serum creatinine measurements indicated the development of mild to moderate renal failure in both diet groups. Phosphaturia was unexpectedly low in all animals that received the STD, indicating relative phosphorus depletion despite the normal dietary phosphorus content. In the HPD CRF group, a decrease in calcemia and a rise in phosphatemia were seen after 12 weeks of CRF, which were more pronounced in animals with higher serum creatinine. Serum iPTH levels were distinctly increased in CRF rats fed a HPD, especially those with more pronounced renal failure. Serum osteocalcin and to a lesser extend tartrate-resistant acid phosphatase and urinary pyridinoline and deoxypyridinoline crosslinks were higher in the CRF animals compared to the shams, particularly in the animals of the HPD group with more pronounced CRF. In both diet groups, the CRF animals had significantly higher amounts of osteoid compared to shams. Only the animals that received a HPD developed distinct histological signs of secondary hyperparathyroidism (sHPTH), that is, an increased bone formation rate, mineral apposition rate, osteoblast perimeter, and eroded perimeter. Again, this effect was most prominent in rats with more severe CRF. In conclusion, data of the present study indicate that in experimental studies using the remnant kidney rat model, both the dietary phosphorus bioavailability and the degree of renal failure in the development of hyperparathyroidism should be considered.
Abstract: Silicon (Si) deficiency in animals results in bone defects. Choline-stabilized orthosilicic acid (ch-OSA) was found to have a high bioavailability compared to other Si supplements. The effect of ch-OSA supplementation was investigated on bone loss in aged ovariectomized (OVX) rats. Female Wistar rats (n = 58, age 9 months) were randomized in three groups. One group was sham-operated (sham, n = 21), and bilateral OVX was performed in the other two groups. OVX rats were supplemented orally with ch-OSA over 30 weeks (OVX1, n = 20; 1 mg Si/kg body weight daily) or used as controls (OVX0, n = 17). The serum Si concentration and the 24-hour urinary Si excretion of supplemented OVX rats was significantly higher compared to sham and OVX controls. Supplementation with ch-OSA significantly but partially reversed the decrease in Ca excretion, which was observed after OVX. The increase in bone turnover in OVX rats tended to be reduced by ch-OSA supplementation. ch-OSA supplementation increased significantly the femoral bone mineral content (BMC) in the distal region and total femoral BMC in OVX rats, whereas lumbar BMC was marginally increased. Femoral BMD was significantly increased at two sites in the distal region in OVX rats supplemented with ch-OSA compared to OVX controls. Total lumbar bone mineral density was marginally increased by ch-OSA supplementation. In conclusion, ch-OSA supplementation partially prevents femoral bone loss in the aged OVX rat model.
Abstract: The mouse mutant Ozzy, originating from an ENU-mutagenesis programme, displays a head bobbing phenotype. We report here that Ozzy mice show a clear deficit in vestibulo-ocular reflex (VOR). Micro-CT scanning of the inner ears showed narrowing and truncations of at least one of the semicircular canals and loss of the ampullae. Frequency-specific auditory-evoked brainstem response (ABR) tests revealed a slight threshold increase in the middle frequency range compared to wild-type littermates. Linkage analysis localised the gene in a 5.5-cM region on chromosome 2. Subsequently, a 499 T-->A missense mutation was identified in Jag1, leading to a substitution of an evolutionary conserved tryptophane (W167R). Mutations in the human homologue of Jag1 cause Alagille syndrome (AGS), an autosomal dominant disorder associated with liver, heart, eye and skeletal abnormalities, accompanied by a characteristic facies. In human patients, it occasionally affects other organ systems like the kidney or the inner ear. Liver disease is the main diagnostic factor for AGS. Ozzy mice showed significantly less intrahepatic bile ducts than wild-type littermates. Thirty-seven percent of Ozzy mice showed heart defects. No eye or vertebral abnormalities could be detected. In conclusion, Ozzy mice show two of the major and one minor characteristic of AGS.
Abstract: BACKGROUND: The Roche CARDIAC proBNP point-of-care (POC) test is the first test intended for the quantitative determination of N-terminal pro-brain natriuretic peptide (NT-proBNP) in whole blood as an aid in the diagnosis of suspected congestive heart failure, in the monitoring of patients with compensated left-ventricular dysfunction and in the risk stratification of patients with acute coronary syndromes. METHODS: A multicentre evaluation was carried out to assess the analytical performance of the POC NT-proBNP test at seven different sites. RESULTS: The majority of all coefficients of variation (CVs) obtained for within-series imprecision using native blood samples was below 10% for both 52 samples measured ten times and for 674 samples measured in duplicate. Using quality control material, the majority of CV values for day-to-day imprecision were below 14% for the low control level and below 13% for the high control level. In method comparisons for four lots of the POC NT-proBNP test with the laboratory reference method (Elecsys proBNP), the slope ranged from 0.93 to 1.10 and the intercept ranged from 1.8 to 6.9. The bias found between venous and arterial blood with the POC NT-proBNP method was < or =5%. All four lots of the POC NT-proBNP test investigated showed excellent agreement, with mean differences of between -5% and +4%. No significant interference was observed with lipaemic blood (triglyceride concentrations up to 6.3 mmol/L), icteric blood (bilirubin concentrations up to 582 micromol/L), haemolytic blood (haemoglobin concentrations up to 62 mg/L), biotin (up to 10 mg/L), rheumatoid factor (up to 42 IU/mL), or with 50 out of 52 standard or cardiological drugs in therapeutic concentrations. With bisoprolol and BNP, somewhat higher bias in the low NT-proBNP concentration range (<175 ng/L) was found. Haematocrit values between 28% and 58% had no influence on the test result. Interference may be caused by human anti-mouse antibodies (HAMA) types 1 and 2. No significant influence on the results with POC NT-proBNP was found using volumes of 140-165 muL. High NT-proBNP concentrations above the measuring range of the POC NT-proBNP test did not lead to false low results due to a potential high-dose hook effect. CONCLUSIONS: The POC NT-proBNP test showed good analytical performance and excellent agreement with the laboratory method. The POC NT-proBNP assay is therefore suitable in the POC setting.
Abstract: BACKGROUND: Patients with impaired renal function can accumulate strontium in the bone, which has been associated with the development of osteomalacia. A causal role for strontium in the development of the disease was presented in chronic renal failure (CRF) rats. Strontium-ranelate has been put forward as a therapeutic agent in the treatment of osteoporosis. Since the target population for strontium treatment consists mainly in postmenopausal osteoporotic women, who may have a reduced renal function, the risk for osteomalacia should be considered. METHODS: To determine the time evolution and reversibility of the strontium-induced mineralization defect, CRF rats were loaded with strontium (2 g/L) (+/- 200 mg/kg/day) during 2, 6, and 12 weeks, followed by a washout period of 0, 2, 4, or 8 weeks. RESULTS: Histologic examination of the bone of the animals treated with strontium revealed signs of osteomalacia already after 2 weeks. Animals that received strontium during 6 and 12 weeks had a significantly higher osteoid perimeter, area and thickness as compared to CRF controls. After 12 weeks, the mineralization was significantly affected, as evidenced by a lower double-labeled surface, mineral apposition and bone formation rate in combination with an increased osteoid maturation time and mineralization lag time. The osteoblast perimeter was significantly lower in the strontium-treated animals. After the washout periods, these effects were reversed and the bone lesions evolved to the values of CRF controls. This went along with an 18% reduction of the bone strontium content. A significant rise in serum alkaline phosphatase (ALP) activity was apparent in the strontium-treated animals as compared to CRF controls. This was not only due to higher levels of the bone ALP but also to those of the liver and the intestinal isoenzymes. Serum parathyroid hormone (PTH) levels decreased during strontium treatment. After cessation of the treatment, the serum ALP activity and PTH concentration reversed to control levels. CONCLUSION: In this study evidence is provided for the rapid development of a mineralization defect in strontium-loaded CRF rats, accompanied by a reduced osteoblast number, reduced PTH synthesis or secretion, and increased serum ALP levels. These effects can be rapidly reversed after withdrawal of the compound.
Abstract: This study was set up to determine the long-term (5 or more years) renal function after HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome during pregnancy and to answer the question whether long-term renal follow-up is necessary. Women with HELLP syndrome were compared with healthy control subjects who delivered their first child during the same period. There was no difference between groups for body mass index, serum and urinary creatinine levels, creatinine clearance, total urinary protein/creatinine ratio, and urinary microalbumin/creatinine ratio. Women who previously had HELLP syndrome had significantly higher diastolic and systolic blood pressures. Women with HELLP syndrome do not need continued renal follow-up, but have higher systolic and diastolic blood pressures, even 5 years after HELLP syndrome.
Abstract: OBJECTIVE: Parietal cell antibodies (PCAs) are found in 20% of type 1 diabetic patients, denoting autoimmune gastritis and pernicious anemia, which may predispose to enterochromaffin-like (ECL) cell hyper/dysplasia and gastric carcinoid tumors. We evaluated whether chromogranin A (CgA), 5-hydroxyindole acetic acid (5-HIAA), and neuron-specific enolase (NSE) contribute to screening for ECL cell hyper/dysplasia. RESEARCH DESIGN AND METHODS: Sera from 93 type 1 diabetic patients (53 men and 40 women, 31 PCA(+) and 62 PCA(-), aged 45 +/- 13 years) were analyzed for PCAs by indirect immunofluorescence and for CgA, NSE, and gastrin by radioimmunoassay. Urinary 5-HIAA was tested by high-performance liquid chromatography. Corpus atrophy and ECL cell proliferation were assessed in gastric biopsies. RESULTS: PCA(+) patients had higher gastrin (P < 0.0001) and CgA levels (P = 0.003) and were more prone to autoimmune gastritis (odds ratio [OR] 17, P < 0.0001) and ECL cell hyper/dysplasia (OR = 23, P = 0.005) than PCA(-) subjects. ECL cell hyper/dysplasia was present in seven PCA(+) patients who showed higher CgA levels (P < 0.0001) than subjects without ECL cell hyper/dysplasia, but NSE and 5-HIAA levels were similar. CgA levels correlated with gastrinemia (r = 0.50, P < 0.0001), PCA titer (r = 0.42, P = 0.001), and 5-HIAA levels (r = 0.38, P = 0.012). Logistic regression identified the CgA level (beta = 0.01, P = 0.027) as an independent risk factor for ECL cell hyper/dysplasia when PCA, CgA, 5-HIAA, NSE, gastrin, sex, and age were tested. Multivariate linear regression demonstrated that CgA level was determined by ECL cell density (r = 0.59, P < 0.0001) and gastrin level (r = 0.67, P = 0.02). One PCA(+) patient with elevated gastrin, CgA, and 5-HIAA levels had a gastric carcinoid tumor. CONCLUSIONS: PCA(+) patients, particularly those with high gastrin and CgA levels, risk developing ECL cell hyper/dysplasia. The determination of CgA, but not NSE and 5-HIAA, may complement histology in evaluating ECL cell mass.
Abstract: AIMS: This study was designed to evaluate the effects of combined endurance/resistance training on NT-proBNP levels in patients with chronic heart failure (CHF). The safety of resistive weight training for patients with CHF is questioned. Possible detrimental effects include an increase in ventricular diastolic pressure and secondary unfavourable remodelling. Circulating levels of the N-terminal fragment of brain natriuretic peptide (NT-proBNP) reflect left ventricular diastolic wall stress and are strongly related to mortality and treatment success in CHF. METHODS AND RESULTS: In this study, 27 consecutive patients with stable CHF and left ventricular ejection fraction (LVEF) <35% were enrolled in a 4 months non-randomized combined endurance/resistance training programme. Blood sampling for measurement of NT-proBNP, functional assessment, cardiopulmonary exercise testing, echocardiography and radionuclide angiography were performed at entry and after 4 months. After 4 months, exercise training caused a significant reduction in circulating concentrations of NT-proBNP (2124+/-397 pg/ml before, 1635+/-304 pg/ml after training, p=0.046, interaction), whereas no changes were observed in an untrained heart failure control group. NYHA functional class (p=0.02, interaction), maximal (peak VO2: p=0.035, interaction; maximal workload: p<0.00001, interaction) and submaximal (workload at anaerobic threshold: p=0.001, interaction; rate-pressure product at anaerobic threshold: p=0.001, interaction) exercise parameters as well as work efficiency (Wattmax/VO2peak: p=0.0001, interaction) were significantly improved. In addition, a decrease in left ventricular end-systolic diameter was observed in the trained heart failure group (p=0.016). CONCLUSION: Four months of combined endurance/resistance training significantly reduced circulating levels of NT-proBNP in patients with CHF, without evidence of adverse remodelling. Exercise training might offer additional non-pharmacological modulation of the activated neurohormonal pathways in the setting of CHF.
Abstract: OBJECTIVE: To report a case of D-lactic acid acidosis owing to massive oral ingestion of propylene glycol. CASE REPORT: A 72-year old man with known congestive failure was admitted to the ICU with encephalopathy. Twelve hours prior to admission he had erroneously ingested a large amount of propylene glycol (PG). The laboratory revealed high anion gap (anion gap = 27 meq/l) acidosis (arterial pH = 7.16) and an increased osmolal gap. Toxicological analysis revealed a low serum propylene glycol level. Biochemical analysis indicated that very high amounts of D-lactic acid (up to 110 mmol/l), but not of the usual type of L-lactic acid, were responsible for the metabolic acidosis. Hemodialysis was initiated and associated with a decline of both the acidosis and D-lactic acid levels. The patient regained conciousness. CONCLUSION: Ingestion of massive doses of propylene glycol, previously not reported as a cause of D-lactic acidosis, should be added to the differential diagnosis of this rare condition.
Abstract: Growing complexity of diagnostic tests, combined with increased workload, stringent laboratory accreditation demands, continuous shortening of turn-around-time and budget restrictions have forced laboratories to automate most of their iterative tasks. Introduction of artificial intelligence by means of expert systems has gained an important place in this automation process. Different parts of clinical laboratory activity can benefit from their implementation and the present project deals with one aspect, namely the clinical interpretation of diagnostic tests. This paper describes how j.MD, a new Java based expert system shell, was used to reprogram the expert system for interpretation of amylase isoenzyme patterns that has been in use for many years in our laboratory, and that was originally programmed in Pro.MD, a Prolog based expert system shell. One of the most important advantages of the j.MD system is its bidirectional link with the laboratory information system. This project shows how expert systems for the interpretation of complex diagnostic tests that demand specific expertise can become an integrated part of the automated clinical chemistry lab.
Abstract: BACKGROUND: Various biochemical markers have been evaluated in dialysis patients for the diagnosis of renal osteodystrophy (ROD). However, their value in predialysis patients with end-stage renal failure (ESRF) is not yet clear. METHODS: Bone histomorphometric evaluation was performed and biochemical markers of bone turnover were determined in serum of an unselected predialysis ESRF population (N = 84). RESULTS: Significant (P < 0.005) differences between the five groups with ROD (ie, normal bone [N = 32], adynamic bone [ABD; N = 19], hyperparathyroidism [N = 8], osteomalacia [OM; N = 10], and mixed lesion [N = 15]) were noted for intact parathyroid hormone, total (TAP) and bone alkaline phosphatase (BAP), osteocalcin (OC), and serum calcium levels. Serum creatinine and (deoxy)pyridinoline levels did not differ between groups. For the diagnosis of ABD, an OC level of 41 microg/L or less (< or =7.0 nmol/L) had a sensitivity of 83% and specificity of 67%. The positive predictive value (PPV) for the population under study was 47%. The combination of an OC level of 41 ng/L or less (< or =7.0 nmol/L) with a BAP level of 23 U/L or less increased the sensitivity, specificity, and PPV to 72%, 89%, and 77%, respectively. ABD and normal bone taken as one group could be detected best by a BAP level of 25 U/L or less and TAP level of 84 U/L or less, showing sensitivities of 72% and 88% and specificities of 76% and 60%, corresponding with PPVs of 89% and 85%, respectively. In the absence of aluminum or strontium exposure, serum calcium level was found to be a useful index for the diagnosis of OM. CONCLUSION: OC, TAP, BAP, and serum calcium levels are useful in the diagnosis of ABD, normal bone, and OM in predialysis patients with ESRF.
Abstract: BACKGROUND: Fifteen to 20% of type 1 diabetic patients exhibit parietal cell antibodies (PCA), which are associated with autoimmune gastritis, hypochlorhydria, iron deficiency and pernicious anaemia. AIM: To examine whether Helicobacter pylori infection could explain the high prevalence of PCA and autoimmune gastropathy in diabetes. If so, H. pylori eradication could prevent autoimmune gastritis. METHODS: In 229 type 1 diabetics (M/F: 135/94; age: 41 +/- 12 years) PCA were measured. H. pylori infection was assessed by serology, urea breath test in all and by histology (updated Sydney system) in 88 subjects. Pentagastrin tests were performed in 42 patients. RESULTS: Sixty-nine patients were PCA-positive. H. pylori infection was present in 72 patients and was negatively associated with HLA-DQA1*0103-B1*0603 (OR=0.12, P=0.015) and positively with DQA1*0501-B1*0201 (OR=1.9, P=0.032). PCA-positivity was linked to HLA-DQA1*0501-B1*0301 (OR=3.9, P=0.017). A link between H. pylori and PCA was observed when PCA-positivity was defined as a titre > or = 1/20 (OR=2.0, P=0.03), but not if > or =1/40 was the cut-off point. PCA-positivity, but not H. pylori infection, was associated with iron deficiency anaemia (OR=2.7, P=0.008), pernicious anaemia (OR= 33.5, P < 0.0001), hypochlorhydria (OR=12.1, P=0.0008) and autoimmune gastritis (OR=12.5, P < 0.0001). CONCLUSIONS: The HLA-bound susceptibility of H. pylori and PCA differed. PCA-positivity but not ongoing H. pylori infection is associated with autoimmune gastritis. Low titres of PCA might reflect H. pylori infection rather than autoimmune gastropathy.
Abstract: Familial amyloidotic polyneuropathy is a systemic amyloidosis with a dismal prognosis for which a surgical treatment exists. Awareness of the clinical characteristics of the disease is critical for early genetic diagnosis and timely referral for liver transplantation. In this report we describe the history of a 49-year-old man in whom non-AA amyloidotic infiltration of the heart and the intestinal tract was diagnosed. Initially, inappropriate identification of the etiology of the disease led to maltreatment.
Abstract: OBJECTIVE: A total of 15-20% of type 1 diabetic patients have parietal cell antibodies (PCAs). PCA+ subjects are at increased risk for iron deficiency anemia and atrophic gastritis. Recently, soluble transferrin receptor (sTfR) levels have proven to be a sensitive indicator for iron deficiency They are, in contrast with ferritin levels, independent of inflammation, liver and hormonal status, and sex. We are the first to evaluate sTfR levels in type 1 diabetes and tested the hypothesis of higher sTfR levels in patients with PCAs and/or autoimmune gastritis. RESEARCH DESIGN AND METHODS: We examined 148 type 1 diabetic patients (85 men and 63 women; 50 were PCA+) and 59 sex- and age-matched control subjects (30 men and 29 women). The main outcome measures were sTfR levels, iron deficiency anemia, and atrophic gastritis. Logistical regression analysis tested risk factors for iron deficiency RESULTS: Iron deficiency was present in 38 subjects. Iron (P<0.0001) and ferritin (P<0.0001) levels but not sTfR levels were lower in women. sTfR levels were similar in diabetic and control subjects but were higher in PCA+ subjects (P = 0.015). In diabetic subjects, iron deficiency anemia was more prevalent in PCA+ than in PCA- patients (odds ratio 3.07, P = 0.013) and was associated with sex (P = 0.0001), age (P = 0.046), and sTfR (P = 0.0008) levels. Atrophic gastritis was present in 15 of 28 PCA+ and in 1 of 11 PCA diabetic subjects (P = 0.014). sTfR levels tended to be higher in patients with atrophic gastritis (P = 0.062). CONCLUSIONS: In type 1 diabetes, sTfR levels can be used to diagnose iron deficiency anemia, which is more prevalent in PCA+ subjects. sTfR levels are higher in PCA+ individuals who are at risk for developing atrophic gastritis.
Abstract: We report on three families with the Gly341Arg ryanodine receptor gene (RYR1) mutation. Thirteen individuals were heterozygote carriers of the Gly341Arg mutation and had clearly positive in vitro contracture tests, indicating malignant hyperthermia susceptibility. Nine Gly341Arg mutation positive individuals from two families had elevated serum creatine kinase (CK) activity at rest (up to six times the normal upper limit). Their clinical and neurological examinations as well as detailed muscle histology were normal. The third family did not show increased CK activity. These findings indicate that the Gly341Arg mutation can be a specific cause of chronically elevated serum CK activity in asymptomatic individuals.
Abstract: Diagnostic enzymology measures the serum or plasma levels of enzymes that were originally located within the cell, or were attached to its plasma membrane with their active sites exposed to the external milieu. The process by which they are released varies under different physiological and pathological conditions. In this way, shedding of hepatocyte plasma membranes is thought to be responsible for the release of liver plasma membrane fragments (LiPMF) into the circulation in metastatic, infiltrative and cholestatic liver diseases. Several membrane-bound enzymes, such as gamma-glutamyltransferase (gamma-GT), alkaline phosphatase (ALP), leucine aminopeptidase (LAP) and 5'-nucleotidase (5'-Nu) are expressed at the surface of the shedded LiPMF. These enzymes are attached to the cell membrane by means of hydrophobic interactions between the anchoring domain of the enzyme and lipid components of the cell membrane, e.g. through a specific glycan phosphatidylinositol (GPI) anchor. There is a striking homology between these LiPMF and the membrane fragments shedded or actively formed by other cells, such as bone matrix vesicles-rich in bone ALP-, membrane fragments of the syncitiotrophoblast-rich in placental ALP-, and membrane fragments present in duodenal fluid-rich in intestinal ALP. With the exception of LiPMF, membrane-bound (Mem-) forms of ALP are only very exceptionally found in human serum. Normally, the soluble (Sol-ALP) dimeric fractions of the enzyme predominate in serum, but liver, bone, placental and intestinal ALP can also be present as GPI-anchor bearing (Anch-) hydrophobic isoforms. Models for the release in the circulation of Mem-, Anch- and Sol-liver and intestinal ALP, involving both plasma membrane-associated GPI-phospholipase-D (GPI-PLD) and liver bile salts are proposed.
Abstract: An immunoradiometric assay (IRMA), involving specific monoclonal antibodies (Ostase, Hybritech) and agarose electrophoresis (Isopal, Beckman), two methods for quantification of serum bone alkaline phosphatase (ALP), a marker for osteoblastic activity, were analytically and clinically compared in 293 patients: 79 with end-stage renal failure treated with hemodialysis and 214 with malignant disease. Acceptable within-assay precision was obtained for the IRMA method: 82.5% of the duplicate determinations had a coefficient of variation (CV) < 5%. Curve fitting characteristics were bad and the sensitivity was better than the one mentioned by the manufacturer. Overall correlation between the two methods was good (r = 0.92), except (a) for low values of bone ALP and (b) in some samples with high total liver ALP activity. Low bone ALP determined with the IRMA (< 5 micrograms/L) was confirmed by electrophoresis (< 22 U/L), but ALP activity determined by electrophoresis to be low (< 22 U/L) was not correlated with the IRMA results. After standardizing our results by computing z-values for bone ALP, delta z (= zostase - zelectrophoresis) was significant correlated with liver ALP activity (r = 0.73, P < 0.0001). We conclude that the IRMA for quantifying bone ALP is acceptable. However, when high values for bone ALP are found with the Ostase method, confirmation by electrophoresis remains mandatory to rule out cross-reactivity with high amounts of liver ALP.
Abstract: BACKGROUND: Adynamic bone disease (ABD) has been described in the current dialysis population to have an unexpectedly high prevalence. Moreover, it is clearly more prevalent in CAPD patients, compared to haemodialysis patients. Recently we demonstrated that both a low (< or = 27 U/1) level of bone alkaline phosphatase (BAP) as determined by an optimized agarose gel electrophoretic technique and a low (< or = 150 pg/ml ) level of iPTH are good markers of ABD with sensitivities of 78.1% and 80.6% and specificities of 86.4% and 76.2% respectively. METHODS: In this study (n = 212), the prevalence of ABD in the European CAPD population was evaluated by means of these biochemical markers. Clinical data on the patients included were recorded at the moment of blood sampling. In patients under CAPD treatment for longer than 9 months, we calculated an index of calcium exposure through PD fluid. RESULTS: In this population with a low exposure to aluminium, the prevalence of ABD as indicated by either a low level of BAP or PTH was 43%. The following risk factors could be identified: advanced age, shorter time on renal replacement therapy, male gender, and high calcium content of PD fluid. The index of calcium exposure was significantly higher in the patients with low BAP and low iPTH levels compared to those with either BAP > or = 27 U/1 or iPTH > 150 pg/ml. The latter finding gives further support to the hypothesis that a high calcium load administered to renal failure patients may lead to 'oversuppressed' parathyroids in ABD. In a subgroup of patients with a high level of BAP associated with a low iPTH level a profile previously shown to be associated in the presence of aluminium overload, significantly higher serum aluminium levels were noted. suggesting that even in patients with low exposure to aluminium, this element still can affect bone metabolism. CONCLUSION: A high prevalence of ABD--as diagnosed by biochemical markers--was observed in the European CAPD population. A number of risk factors could be put forward. The aetiology and pathogenesis of this type of renal osteodystrophy remain to be elucidated, but appear, however, to be multifactorial.
Abstract: Membrane-bound liver alkaline phosphatase (Mem-LiALP, EC 3.1.3.1) is a high-molecular-mass liver alkaline phosphatase (ALP) present in metastatic, infiltrative and cholestatic liver disease. Shedding of hepatocyte plasma membrane fragments (LiPMF) is thought to be responsible for the appearance of Mem-LiALP in the circulation. Several other membrane-bound enzymes, such as gamma-glutamyltransferase (gamma-GT), leucine aminopeptidase (LAP), and 5'-nucleotidase (5'-Nu) are present in the membrane of the shedded LiPMF. By means of immunohistochemical and immunoassay procedures, we presently show that AD-1, a specific monoclonal antibody originally produced against Mem-LiALP, reacts with LAP, a constituent of the human liver plasma membrane. Using AD-1 as an immunosorbant, we isolated circulating LiPMF from cholestatic sera to a high level of purity and separated it from other high-molecular-mass material, such as liver ALP or similar lipoprotein-X complexes. These purified membrane fragments retained their biochemical characteristics. Glycosyl-phosphatidylinositol anchor bearing liver ALP (Anch-LiALP) could be released from the LiPMF by Triton X-100. Whereas ALP was released upon treatment of AD-1 purified LiPMF with phospholipase C, phospholipase D only cleaved the glycosyl-phosphatidylinositol anchor following detergent solubilization of the enzyme. Serum LiPMF from patients with different kinds of cholestatic liver disease were bound onto AD-1 coated nitrocellulose disks and the activity of four membrane-bound enzymes (LAP, ALP, 5'Nu, gamma-GT) was analyzed. A considerable interindividual variation of enzyme activities was observed, suggesting some heterogeneity in the membrane composition of these fragments.
Abstract: BACKGROUND. Adynamic bone disease was recently described to be increasingly prevalent in the dialysis population. At present the diagnosis of this type of renal osteodystrophy can only be made by bone histomorphometry. We assessed the value of different biochemical serum markers in the diagnosis of adynamic bone disease. METHODS. In 103 haemodialysis patients a bone biopsy was performed after double tetracycline labelling, and the serum levels of intact PTH, osteocalcin, and the bone isoenzyme of alkaline phosphatase were determined. Bone alkaline phosphatase was measured by an optimized agarose gel electrophoretic method, recently shown to have a high accuracy, precision and reproducibility, also in the lower range. RESULTS. In 38 (37%) of the patients the diagnosis of adynamic bone disease was histologically established. Constructing receiver operator curves optimal cut-off levels for the diagnosis of adynamic bone disease were determined, being <=27 U/litre for the bone isoenzyme of alkaline phosphatase, <=14 microg/litre for osteocalcin and <=150 pg/ml for intact PTH. Concentrations of bone alkaline phosphatase or intact PTH below these cut-off levels, were shown to be the best performing tests in the detection of adynamic bone disease as indicated by a sensitivity of 78.1 and 80.6% and a specificity of 86.4 and 76.2% respectively. Applying Bayes' theorema, it was calculated that in the current haemodialysis population in which a prevalence of adynamic bone disease up to 35% has been described, the positive predictive values for the proposed cut-off values are 75% for bone alkaline phosphatase, 65% for intact PTH and 55% for osteocalcin. Moreover, in this population, levels of bone alkaline phosphatase and intact PTH below the optimal cut-off excluded hyperparathyroid bone disease. CONCLUSION. In view of the relative easy and accurate methodology for bone alkaline phosphatase determination, the closer physiological link with osteoblast function and the lesser expense for its determination we suggest that this marker is a useful tool in the non-invasive diagnosis of the adynamic type of bone disease in the individual patient.
Abstract: Circulating liver plasma membrane fragments (LPMF) were purified from human serum by means of a monoclonal antileucine aminopeptidase antibody, AD-1. This was done by immunoaffinity chromatography or by incubating the sera with AD-1-coated nitrocellulose disks. Alkaline phosphatase (ALP, EC 3.1.3.1) is bound to these LPMF through a glycosylphosphatidylinositol (GPI) anchor and is referred to as membrane-bound liver ALP (Mem-LiALP). Low concentrations of Triton X-100 or high bile salt concentrations released GPI anchor-bearing LiALP (Anch-LiALP) from purified LPMF; once released, Anch-LiALP was slowly and progressively converted to hydrophilic dimeric LiALP [soluble LiALP (Sol-LiALP)], free from its GPI anchor. Low levels of GPI-specific phospholipase D (GPI-PLD) activity were measured in the pure LPMF. Apparently, this membrane-associated GPI-PLD was released by the action of detergents and contributed to the spontaneous conversion of Anch-LiALP to Sol-LiALP. In the absence of detergents, GPI-PLD had little effect on Mem-LiALP, both in purified form as well as in serum. In vitro, isolated Anch-LiALP was converted to Sol-LiALP by both GPI-specific phospholipase C and GPI-PLD. Sol-LiALP in serum, however, appeared to be the product of GPI-PLD activity only. Five- to tenfold higher concentrations of Triton X-100 were needed to release Anch-LiALP from LPMF in serum, compared with those required in a solution of purified LPMF. In serum, as well as in purified conditions, only a small range of detergent of bile salt concentrations permitted the conversion of Mem-LiALP to Sol-LiALP. A model is proposed for the release in the circulation of Mem-LiALP, Anch-LiALP, and Sol-LiALP, involving both LPMF-associated GPI-PLD and liver sinusoid bile salts.
Abstract: Agarose electrophoresis (Isopal, Beckman) and an immunoradiometric assay (IRMA) involving specific monoclonal antibodies (Ostase, Hybritech), two methods for the quantification of serum bone alkaline phosphatase (ALP, EC 3.1.3.1), a marker of osteoblastic activity, were compared in 293 patients: 79 with end-stage renal failure treated with hemodialysis and 214 with malignant disease. Overall correlation between the two methods was good (r = 0.92), except (a) for low values of bone ALP and (b) in some samples with high total liver ALP activity--both due to considerable cross-reactivity of the anti-bone ALP antibodies of the Ostase kit with liver ALP. This interference was not constant and was not evenly distributed across all concentrations of bone ALP. Low bone ALP determined with the IRMA (< or = 5 micrograms/L) was confirmed by electrophoresis (< or = 21 U/L), but bone ALP activity determined by electrophoresis to be low (< or = 21 U/L) was not correlated with the IRMA results. After standardizing our results by computing z-values for bone ALP, delta z (= zOstase - zIsopal) was significantly correlated with liver ALP activity (r = 0.73, P < 0.0001). We conclude that the IRMA for quantifying bone ALP is acceptable as a screening method. However, when high values for bone ALP are found with the Ostase method, confirmation by electrophoresis remains mandatory to rule out cross-reactivity with high amounts of liver ALP. For detecting low bone ALP activities, electrophoresis remains the method of choice.
Abstract: Alkaline phosphatase (ALP, EC 3.1.3.1) is a membrane-bound metalloenzyme that consists of a group of true isoenzymes, all glycoproteins, encoded for by at least four different gene loci: tissue-nonspecific, intestinal, placental, and germ-cell ALP. Through posttranslational modifications of the tissue-nonspecific gene, for example, through differences in carbohydrate composition, bone and liver ALP are formed. Nowadays, most commercially available methods for separating or measuring ALP isoenzymes are easy to perform and sensitive and allow for reproducible and quantitative results. As more isoenzymes and isoforms have been characterized, confusion has arisen due to the many different names they were given. For the sake of simplicity and because of structural analogies, we propose an alternative nomenclature for the ALP isoenzymes and isoforms based on their structural characteristics: soluble, dimeric (Sol), anchor-bearing (Anch), and membrane-bound (Mem) liver, bone, intestinal, and placental ALP. Together with lipoprotein-bound liver ALP and immunoglobulin-bound ALP, these names largely fit the many forms of ALP one can encounter in human serum and tissues. The clinically relevant isoenzymes are sol-liver, Mem-liver, lipoprotein-bound liver, and Sol-intestinal ALP in liver diseases, and Sol-bone and Anch-bone ALP in bone diseases. Many different isoenzyme patterns can be found in malignancies and renal diseases. This test provides the clinician with valuable information for diagnostic purposes as well as for follow-up of patients and monitoring of treatment. However, ALP isoenzyme determination will only provide clinically useful information if the patterns are correctly interpreted. In this respect, care should be taken to use the proper reference ranges, taking into account the age and sex of the patient. A normal total ALP activity does not rule out the presence of an abnormal isoenzyme pattern, particularly in children. Separating ALP into its isoenzymes adds considerable value to the mere assay of total ALP activity.
Abstract: Human intestinal alkaline phosphatase (IAP) can be released by the enterocyte into duodenal fluid as a mixture of three isoforms. A proportion of the enzyme is associated with triple-layered membrane vesicles (vesicular IAP). Although, occasionally, free hydrophilic IAP dimers are present, the remaining enzyme usually consists of a mixture of hydrophobic IAP dimers and more complex hydrophobic IAP structures of larger size, both entities being identified as "intestinal variant" alkaline phosphatase (VAR IAP). The hydrophobicity of VAR IAP stems exclusively from its attached glycosyl-phosphatidylinositol (GPI) anchor. Both vesicular IAP and VAR IAP are converted to hydrophilic enzyme upon removal of the GPI tail by phospholipase D (PLD) present in duodenal fluid. The IAP released into the vascular bed consists mainly of VAR IAP; vesicular IAP is absent. The enzyme characteristics of VAR IAP partially purified from duodenal fluid and from serum are identical. In plasma, VAR IAP appears to associate with (lipo)protein complexes and is thus protected from further degradation by plasma PLD. Such complex formation may explain why, in the serum of a healthy reference population, VAR IAP was more abundant than hydrophilic dimeric IAP.
Abstract: Early treatment of patients with malignant disease and liver or bone metastasis may increase their survival time. We have used the activity patterns of liver and bone isoenzymes of alkaline phosphatase (ALP), separated by agarose gel electrophoresis, to detect early metastasis. We studied ALP isoenzyme patterns in a background population of 101 patients with no evidence of any disease that might influence this pattern; a healthy reference population (n = 330); and the following three groups of patients: 143 with malignant disease, 47 with nonmalignant liver disease, and 22 with nonmalignant bone disease. Cutoff and predictive values of liver ALP, high-molecular-mass (high-M(r)) ALP, and bone ALP were established for detecting liver and bone metastasis. The positive predictive value of liver and high-M(r) ALP was higher than that of total ALP in detecting liver metastasis, but liver and high-M(r) ALP did not enable us to differentiate between malignant and nonmalignant liver disease. Total ALP activity was of slightly more value than liver and high-M(r) ALP in enabling us to rule out liver metastasis. From bone ALP activity we could not distinguish between nonmalignant bone disease and bone metastasis. The negative predictive value of bone ALP in the diagnosis of bone metastasis was low, but its positive predictive value was high and superior to that of total ALP.
Abstract: The diagnostic value of serum amylase determination for pancreatic disease has been questioned due to its lack of specificity. Several methods have been developed to separate the tissue-unspecific salivary fractions from the tissue-specific pancreatic fractions. Agarose or cellulose acetate gel electrophoresis are most suitable for clinical practice. The isoamylase patterns were studied by agarose electrophoresis in 55 patients with known pancreatic diseases (acute pancreatitis, pancreatic pseudocysts, exocrine pancreatic insufficiency and pancreatic carcinoma). Increased P-type isoamylase seems to be more sensitive than total amylase in diagnosing acute pancreatitis, while identification of the minor isoamylase P3 is more specific and could have a prognostic value. Detection of low P-type isoamylase levels is an easy method to diagnose exocrine pancreatic insufficiency. Furthermore, a group of patients with pancreatic disease (Pa), was compared with a group of patients with biliary disease without clinical evidence of pancreatic involvement (Bi), and patients with abdominal pain, without evidence of biliary or pancreatic disease (Ab). More than half of the Bi patients presented with abnormal P isoenzyme patterns, whereas 72% of the Ab patients had a normal pattern. Only P3 could distinguish between the Bi and Ab group. This might point to pancreatic involvement in patients presenting with biliary disease, only detected by isoamylase analysis.
Abstract: We separated isoenzymes of alkaline phosphatase (ALP; EC 3.1.3.1) in 1383 sera of normal individuals (ages 4-65 years) by agarose electrophoresis with the Isopal system (Analis). As expected, the predominant isoenzyme in children was of bone origin, and almost all (99%) of the children had low activities of a second bone fraction, "bone variant" ALP. The "bone variant" disappeared after age 17 in girls and after age 20 in boys. The highest (median) bone ALP activity was reached at age 9 to 10 in girls and at age 13 to 14 in boys, followed by a gradual decline in girls and a steep decline in boys. During adulthood, activity of the bone fraction was constant and no significant differences were observed between sexes, neither for bone nor for liver ALP activity. The latter remained almost unchanged throughout life. We observed no high-Mr ALP activity in children, whereas sera from 60% of the adults contained low activities of high-Mr ALP. Intestinal ALP (soluble form) and "intestinal variant" ALP (hydrophobic form) were frequently present, in 21% and 37% of all samples, respectively. No significant differences were observed between age groups and sexes for the intestinal isoenzymes.
Abstract: Human long-term bone marrow cultures (HLTBMCs) were established with bone marrow (BM) collected from five patients with myelomatous disorders (four with multiple myeloma, one with plasma cell leukemia). In all cases, up to at least 6 weeks of culture, there was a persistence of the monoclonal plasma cell population in the adherent layer of the culture. In some cultures proliferating plasma cells could be demonstrated by the Ki-67 monoclonal antibody. In all instances a paraprotein could be shown in the conditioned medium. This study demonstrates that malignant plasma cells, in analogy to their normal counterparts, have an affinity for the BM stroma and suggests that their long-term survival might be enhanced by their interaction with it.
Abstract: A modified agarose electrophoretic system for the separation of alkaline phosphatase (ALP, EC 3.1.3.1) isoenzymes is described. Bone, liver, high-molecular-mass, and intestinal ALP are separated with high reproducibility. The sensitivity of the agarose system is superior to cellulose acetate in detecting high-Mr ALP. Correlation is good between bone ALP fractions scanned before and after treatment with neuraminidase. Immunoglobulin-bound ALPs, the ALP-lipoprotein-X complex, and the additional ALP fraction observed in transient hyperphosphatasemia in children are detected by their peculiar electrophoretic mobility in the proposed system. Approximately 25% of the samples contained an additional fraction of intestinal-type ALP, as evidenced by neuraminidase treatment and use of polyclonal and monoclonal antibodies. Because the electrophoretic mobilities of this "intestinal variant" and of some immunoglobulin-bound ALP fractions are identical to those of bone and intestinal ALP, respectively, treatment of the samples with a polyclonal antibody that reacts with intestinal ALP is advised.
