Rob NM Weijers

Abstract: ABSTRACT: BACKGROUND: We examined the effects of the R325W mutation on the three-dimensional (3D) structure of the beta-cell-specific Zn2+ (zinc) transporter ZnT-8. METHODS: A model of the C-terminal domain of the human ZnT-8 protein was generated by homology modeling based on the known crystal structure of the Escherichia coli (E. coli) zinc transporter YiiP at 3.8 A resolution. RESULTS: The homodimer ZnT-8 protein structure exists as a Y-shaped architecture with Arg325 located at the ultimate bottom of this motif at approximately 13.5 A from the transmembrane domain juncture. The C-terminal domain sequences of the human ZnT-8 protein and the E. coli zinc transporter YiiP share 12.3% identical and 39.5% homologous residues resulting in an overall homology of 51.8%. Validation statistics of the homology model showed a reasonable quality of the model. The C-terminal domain exhibited an alphabetabetaalphabeta fold with Arg325 as the penultimate N-terminal residue of the alpha2-helix. The side chains of both Arg325 and Trp325 point away from the interface with the other monomer, whereas the epsilon-NH3+ group of Arg325 is predicted to form an ionic interaction with the beta-COO- group of Asp326 as well as Asp295. An amino acid alignment of the beta2-alpha2 C-terminal loop domain revealed a variety of neutral amino acids at position 325 of different ZnT-8 proteins. CONCLUSIONS: Our validated homology models predict that both Arg325 and Trp325, amino acids with a helix-forming behavior, and penultimate N-terminal residues in the alpha2-helix of the C-terminal domain, are shielded by the planar surface of the three cytoplasmic beta-strands and hence unable to affect the sensing capacity of the C-terminal domain. Moreover, the amino acid residue at position 325 is too far removed from the docking and transporter parts of ZnT-8 to affect their local protein conformations. These data indicate that the inherited R325W abnormality in SLC30A8 may be tolerated and results in adequate zinc transfer to the correct sites in the pancreatic islet cells and are consistent with the observation that the SLC30A8 gene variant R325W has a low predicted value for future type 2 diabetes at population-based level.

Abstract: Reduced insulin sensitivity plays a role in the early pathogenesis of type 2 diabetes, and defects in insulin secretion by pancreatic beta-cells are instrumental in hyperglycemic progression. There is strong evidence that genetic factors play an important role in both of these components. Several of the single nucleotide polymorphisms (SNPs) of genes associated with an increased risk of type 2 diabetes are hypothesized to influence beta-cell function. The aim of the present study was to describe the function of the latter genes, to analyze the implications of the SNP positions within or near these genes, and to evaluate the suggested primary role of pancreatic beta-cells in the etiology of type 2 diabetes.

Abstract: Verondersteld wordt dat op moleculair niveau een groot aantal opeenvolgende reacties betrokken is bij het ziekteproces van diabetes type 2. Voorbeelden hiervan zijn de ontwikkeling van de ß-cel, de signalering via de insulinereceptor, de synthese en het gebruik van koolhydraten, het mitochondrieel metabolisme, de oxidatie van vetzuren, de signalering via cytokinen en het ontstaan van hyperlipidemie. Onderzoek naar gecoördineerde veranderingen in de expressie van groepen van functioneel gerelateerde genen, heeft een verband aangetoond tussen diabetes type 2 en aangeboren afwijkingen in mitochondrieel DNA.

Abstract: Het artikel over diabetes in C2W life sciences 12 legt veel nadruk op insulineresistentie. Opname van glucose kan echter ook zonder insuline, betoogt klinisch chemicus Rob Weijers

Abstract: BACKGROUND: We examined the pathogenesis of gestational diabetes mellitus (GDM) in a large Dutch multiethnic cohort. METHODS: We used a 2-step testing procedure to stratify 2031 consecutive pregnant women into 4 groups according to American Diabetes Association criteria: (a) normal glucose tolerance (NGT), (b) mild gestational hyperglycemia (MGH), (c) GDM without early postpartum diabetes within 6 months of delivery (GDM1), and (d) GDM with early postpartum diabetes (GDM2). Antepartum and postpartum clinical characteristics and measures of glucose tolerance were documented. RESULTS: Overall, 1627 women had NGT, 237 had MGH, 156 had GDM1, and 11 had GDM2. Prepregnancy body mass index values progressively increased from NGT to MGH to GDM1. The fasting plasma glucose concentration, the 100-g oral glucose tolerance test (OGTT) area under the curve, and the mean glucose concentration during the OGTT all increased progressively among the 4 groups. The fasting C-peptide concentration displayed an inverted-U pattern, with a maximum at a mean plasma glucose concentration during the OGTT of 9.6 mmol/L in the transition from GDM1 to GDM2. The fasting C-peptide/glucose concentration ratio decreased by 42% in GDM patients compared with NGT patients, whereas the ratios in MGH and NGT women were similar. CONCLUSIONS: Progressive metabolic derangement of glucose tolerance 1st detected during pregnancy mimics the pathogenesis of type 2 diabetes. In addition, our results imply an impaired basal glucose effectiveness in the early prediabetic state. To explain the parallel in both metabolic derangements, we postulate that GDM, like type 2 diabetes, is attributable to the same inherited mitochondrial dysfunction.

Abstract: We examined the clinical usefulness of antepartum clinical characteristics, along with measures of glucose tolerance, in Dutch multiethnic women with gestational diabetes mellitus (GDM) for their ability to predict type 2 diabetes within 6 months of delivery (early postpartum diabetes). The present study comprised a cross-sectional 5-year investigation (1998-2003) of a consecutive series of 168 women with GDM identified by a two-stage protocol at 16-33 weeks of gestation. The following data were collected for all women: age and gestational age at entry into the study; prepregnancy body mass index (BMI); ethnicity; obstetric and clinical history, including the onset of early postpartum diabetes; pregnancy outcome; level of fasting C-peptide; and glycemic parameters of 50-g 1-h glucose challenge test and 100-g 3-h oral glucose tolerance test (diagnostic OGTT). We used receiver operating characteristic (ROC) curve analysis to test the clinical usefulness of the glycemic parameters. A total of 11 women (6.5%) developed early postpartum diabetes. Apart from family history of diabetes (p = 0.052), anthropometric, maternal, and neonatal clinical parameters showed no association with early postpartum diabetes in univariate analyses. The level of fasting glucose, and both the glucose challenge test and diagnostic OGTT post-load glucose levels and glucose areas were associated with early postpartum diabetes. ROC curve analysis identifiedall three glucose challenge-test parameters, including fasting glucose concentration, as poor diagnostic tests, with a positive predictive value of approximately 22%, whereas the positive predictive value associated with the area under the diagnostic OGTT curve increased progressively over monitoring time from 20.6% to 100%. Using a 3-h OGTT glucose area threshold of 35.7 mmol.h/L resulted in 100% sensitivity and 100% specificity, identifying the 11 women who developed early postpartum diabetes. In summary, we can conclude from the present analysis that early postpartum diabetes is rare in GDM women (6.5%), and that the clinical usefulness of the total area under the diagnostic 3-h OGTT is superior to all other glycemic parameters for detecting early postpartum diabetes.

Abstract: De laatste jaren heeft de wetenschap op het terrein van type-2-diabetes niet stilgestaan. Onder meer is belangrijke progressie geboekt op het gebied de pathofysiologie van de ziekte. Zo is gebleken dat hyperinsulinemie bij de overgang van prediabetes naar diabetes gepaard gaat met depositie van amyloïd in de pancreas. Welke implicaties hebben deze nieuwe inzichten voor de diagnostiek en behandeling?

Abstract: Als leidraad voor de diagnostiek, behandeling en begeleiding van de diabetespatiënten door de huisarts heeft het Nederlandse Huisartsen Genootschap in 1998 een herziene standaard diabetes mellitus type 2 geformuleerd. Sinds 1998 heeft de wetenschap op het terrein van type-2-diabetes evenwel niet stil gestaan. Onder meer is belangrijke progressie geboekt op het gebied van de pathofysiologie van de ziekte. Zo is gebleken dat de hyperinsulinemie bij de overgang van prediabetes naar diabetes gepaard gaat met depositie van amyloïd in de pancreas. Welke implicaties hebben deze nieuwe inzichten voor de diagnostiek en behandeling?

Abstract: OBJECTIVE: The purpose of this study was to identify independent determinants of mild gestational hyperglycemia (MGH) and gestational diabetes mellitus (GDM) and to assess the correlation between fasting glucose and C-peptide levels among control, MGH, and GDM women. RESEARCH DESIGN AND METHODS: A total of 1,022 consecutive women were evaluated with a 1-h 50-g glucose challenge test (GCT) at between 16 and 33 weeks of gestation. Women with a capillary whole-blood glucose > or =7.8 mmol/l in the GCT underwent a 3-h 100-g oral glucose tolerance test (OGTT). On the basis of a positive GCT, the women with a positive OGTT were classified as GDM, whereas the women with a negative OGTT were classified as MGH. The following data were collected for all women: age, prepregnancy BMI, ethnicity, clinical and obstetric history, pregnancy outcome, and C-peptide level. RESULTS: A total of 813 women (79.6%) were normal, 138 (13.5%) had MGH, and 71 (6.9%) had GDM. There was a stepwise significant increase in mean fasting glucose (3.6 +/- 0.4, 3.9 +/- 0.4, and 4.7 +/- 0.7 mmol/l, respectively) and C-peptide level (0.60 [0.1-2.4], 0.86 [0.3-2.0], and 1.00 [0.5-1.6] nmol/l, respectively) among the three diagnostic groups. Maternal age, non-Caucasian ethnicity, and prepregnancy BMI were associated with GDM, whereas only maternal age and prepregnancy BMI were associated with MGH. A positive correlation between levels of fasting glucose and C-peptide was found in control women (r = 0.39 [95% CI 0.31-0.46]). A similar result was seen in MGH women (r = 0.38 [95% CI 0.23-0.52]), whereas the correlation between fasting glucose and C-peptide was nearly lost in GDM women (r = 0.14 [CI -0.09 to 0.36]). The fasting C-peptide-to-glucose ratio was reduced by 60% in GDM patients versus control subjects and MGH patients (0.41 +/- 0.25 vs. 0.70 +/- 0.20 and 0.73 +/- 0.23, P < 0.001). CONCLUSIONS: Of the well-known independent determinants of GDM, only maternal age and prepregnancy BMI were associated with MGH. It appears that additional factors promoting loss of beta-cell function distinguish MGH from GDM. One of these factors appears to be ethnicity.

Abstract: Holland was one of the first countries to be involved in diabetes quality assurance initiatives and was represented on the original working groups that defined the gold standards for diabetes care in the late 1980s. In the Netherlands, the focus is on providing care for type 2 diabetic patients via GPs, supplementing this with structured monitoring at quality centres, such as the OLVG. The Camit software, also based on the basic information sheet, is a key tool for measuring and managing diabetes. By the end of2001, up to 25 hospitals in the Netherlands plus GPs will be participating in the benchmarking. Family physicians are now much more aware of diabetes care issues than they were, and I know that the benchmark data have started some very good dialogue between physicians, and between physicians and their patients.

Abstract: Background: The aim of this study was to identify the independent determinants of diabetic retinopathy (RET) and microalbuminuria (MA) and to assess the time-dependency of the association of RET with MA. Methods: In 668 out-patients with type 2 diabetes, RET was assessed by stereoscopic fundoscopy and by measuring the level of MA in untimed, triplicate urine collections on at least two and four separate visits, respectively, during a period of at least 24 months. RET was defined as RET of any type and MA as a urinary albumin-to-creatinine ratio (ACR) between 2 and 30 mg/mmol. Multiple logistic regression analysis was used to determine odds ratios (OR) and 95% confidence intervals (CI). The extent of the association (OR(assoc)) was estimated by the odds that a patient with RET has MA divided by the odds that a patient without RET has MA. Results: Common determinants of RET and MA were: systolic BP, HbA(1c), and triglycerides. Age, non-Caucasian ethnicity, and RET were associated with MA, whereas duration of diabetes and ACR were associated with RET. We estimated an overall OR(assoc) of 2.36 (95% CI, 1.72-3.24). The time-dependency of OR(assoc) showed a hyperbolically shaped curve, reaching a maximum value of 2.5 at 9.8 years after the diagnosis of type 2 diabetes. Conclusions: Our study, which supports what is currently known about independent determinants of diabetic RET and MA, suggests a drastic increase in clustering of RET and MA over the first 5 years before the diagnosis of type 2 diabetes.

Abstract: To implement the St. Vincent Declaration Action Programme, initiatives have been started in the quality development of diabetes care across Europe. The main elements of the quality development cycle implemented in our hospital were realized by the use of the software package 'CamitPro' and participation in 'the DiabCare Q-net NL' network. From 1997 to 1999, 955, 1468, and 1624 patients with diabetes mellitus type 2 were integrated in the DiabCare Q-net NL network by using 'CamitPro' software, respectively. These patients showed substantial improvement in all of the clinical measures monitored. In addition, there was a drop in HbA1c level. From 1997 to 1999, an HbA1c interval of 5.1%-8.3% (mean up to mean +4SD) was recorded for 66%, 76% and 81% of the patients, respectively. An extension of the use of the software to a pan-European level should markedly improve diabetes care throughout the community.

Abstract: Zeeburg', a multiethnic town borough in the Amsterdam-East region, has one of the city's highest rates of immigrants. In the total population of 19,825 Surinam (mainly Creole), Turkish, Moroccan, and Dutch adults the prevalence of known type 2 diabetes in 1994 and of gestational diabetes mellitus (GDM) between January 1992 and January 1997 was investigated. Based on World Health Organization (WHO) criteria of 1985, the age-standardized prevalence of type 2 diabetes was similar in men (6.4%; 95% confidence interval [CI]: 5.6-7.2) and women (6.4%: 95% CI: 5.8-7.0) for all ethnic groups combined. However, the age- and sex-standardized prevalence of type 2 diabetes was significantly greater in the non-Dutch inhabitants than in the Dutch inhabitants (17.3% [95% CI: 12.9-21.6] in Surinam inhabitants, 10.9% [95% CI: 9.7-12.2] in Turkish inhabitants, 12.4% [95% CI: 9.7-15.0] in Moroccan inhabitants, and 3.6% [95% CI: 3.2-3.9] in Dutch inhabitants). The odds ratios for type 2 diabetes for the separate immigrant groups relative to the Dutch group were 5.88 (95% CI: 4.54-7.69) for Surinam inhabitants, 4.00 (95% CI: 2.86-5.55) for Turkish inhabitants, and 4.17 (95% CI: 3.03-5.55) for Moroccan inhabitants. GDM was present in 2.59% of women of non-Dutch origin compared with 0.62% of women of Dutch origin. A significant positive association was found between the non-Dutch origin and the occurrence of GDM (chi2 = 6.7; p < 0.01). The study highlights a high prevalence of known type 2 diabetes and GDM in the immigrant inhabitants and emphasizes that appropriate interventions are necessarily with implications for health targets and capitation based budgets.

Abstract: The purpose of this study was to investigate the effect of ethnicity on the development of diabetic retinopathy and nephropathy as markers for microvascular complications and of angina pectoris as a marker for macrovascular complications. We evaluated data from 1124 patients with non-insulin-dependent diabetes mellitus (NIDDM) of Caucasian, Mongoloid, Asian, Armenian, Northern African and Negroid origin who were referred between January 1993 and December 1994. Logistic regression analyses showed that the occurrence of microvascular complications was significantly associated with duration of NIDDM. In addition, retinopathy was significantly associated with glycated haemoglobin A1c (HbA1c) and nephropathy with triglycerides (P < 0.05 and P < < 0.001 respectively). Northern African origin was associated with retinopathy (P < 0.05) and Asian origin with nephropathy (P < 0.005). Macrovascular complication was associated with age and triglyceride level (P < 0.001 and P < 0.05 respectively). Northern African and Negroid ethnicity exclusively did not show a gradual increase in the risk for angina pectoris with increasing age. Moreover, a negative association between Northern African as well as Negroid ethnicity and macrovascular complication was observed (P = 0.05 and P < 0.05 respectively). In support of these observations, we found a favourable lipid profile in both mentioned groups. In summary, we have shown that, in patients with NIDDM, ethnicity is associated with macrovascular complications and duration of the disease with microvascular complications.

Abstract: The objective of this study was to investigate whether reduction in hypertriglyceridaemia is associated with a slower rate of progression of microalbuminuria in patients with non-insulin-dependent diabetes mellitus (NIDDM). Fifteen normotensive NIDDM patients with hypertriglyceridaemia (> 2.5 mmol L-1) and microalbuminuria were randomly selected to receive either placebo (eight patients) or gemfibrozil 600 mg b.i.d. (seven patients). Progression of microalbuminuria was assessed during a 12-month follow-up period with measurements, consisting of blood tests and triplicate 24-h urine collections, at 1, 3, 6, 9 and 12 months. All but one patient in the treatment group showed a favourable response (> or = 20% reduction) of hypertriglyceridaemia to gemfibrozil. One patient in the placebo group showed a spontaneous reduction in triglyceride levels. Progression of microalbuminuria was lower, although not statistically significantly so, in the treatment group (36%) than in the placebo group (65%). In the group with > or = 20% reduction in triglyceride levels, progression of MA was significantly lower than in the group with stable or increasing triglyceride levels (+1%, range -56% to +49% vs. +97%, range -35% to +202% respectively) (P = 0.03). Continued follow-up data of patients switching from placebo to gemfibrozil after the trial further support the role of serum triglyceride reduction in stabilizing albumin excretion. In conclusion, the results indicate that, in microalbuminuric NIDDM patients, effective treatment of dyslipidaemia could be associated with stabilization of urinary albumin excretion.

Abstract: OBJECTIVE: To assess the degree of interindividual variation in the rate of progression of microalbuminuria and to identify determinants of progression of microalbuminuria in patients with NIDDM. RESEARCH DESIGN AND METHODS: In a prospective cohort study, 58 microalbuminuric NIDDM patients were followed for a period of at least 24 months. During this period, the level of microalbuminuria in these patients was assessed in triplicate 24-h urine samples on at least four separate visits. All patients had stable metabolic control and controlled blood pressure during follow-up. Microalbuminuria was defined as an albumin-to-creatinine ratio in 24-h urine of between 3 and 30 mg/mmol. The individual rates of progression of microalbuminuria were calculated from linear regression analysis. At baseline, the following data were collected for all patients: age, sex, ethnicity, time since diagnosis of NIDDM, smoking habits, drug use, blood pressure, BMI, HbA1c, serum creatinine, cholesterol, triglyceride, and HDL cholesterol concentrations. RESULTS: Microalbuminuria was found to progress linearly in time. Considerable differences in rates of progression of microalbuminuria were found, the absolute yearly change in albumin-to-creatinine ratio ranging from -5.2 to 12.9 mg/mmol. In bivariate analyses, serum triglyceride concentration, use of ACE inhibitors, mean arterial blood pressure, HDL cholesterol, and time since diagnosis of NIDDM correlated with progression of microalbuminuria (P < or = 0.05). In stepwise multiple regression analysis, a high triglyceride-to-HDL cholesterol ratio at baseline (P = 0.006) and the use of ACE inhibitors (P = 0.007) were identified as the only independent predictors of progression of microalbuminuria. CONCLUSIONS: The rate of progression of microalbuminuria in NIDDM differs considerably between subjects. Diabetic dyslipidemia (high serum triglyceride and low HDL cholesterol) is a predictor of more rapid progression of microalbuminuria in patients with well-controlled blood pressure.

Abstract: We investigated the anticoagulating and heparin-neutralizing properties of protamine and polybrene (hexadimethrine bromide), using the endogenous thrombin potential (ETP) as the parameter to access plasma coagulability. The hypocoagulability induced by high doses of heparin (3 IU/ml) could be reversed by addition of protamine to a very limited extent only. Polybrene on the other hand did neutralize heparin at the equivalent concentration and a two-fold excess did not influence the ETP parameters. In vivo neutralization of high-dose heparin with protamine should therefore be reconsidered. Our experiments suggest polybrene to be superior over protamine with respect to neutralization of high doses of heparin.

Abstract: Macro enzymes, i.e. complexes of normal (iso-)enzymes with an immunoglobulin, may be due to immunological cross-reactions evoked by specific viral antigenic determinants that are homologous to regions in the target enzymes. A search of the National Biomedical Research Foundation protein databank with the amino-acid sequence of human pancreatic amylase revealed a marked homology with a fragment of the yellow fever virus major envelope protein E: i.e. an overall identity of 19.7 per cent and a high degree (40.9 per cent) of conservative amino-acid substitutions over 119 amino acids. At each identical position, the corresponding residue of Taka amylase A was examined by three-dimensional structure analysis, to determine whether the position is likely to be buried or exposed. The existence of a site (epitope) on amylase recognized by an anti-amylase antibody is discussed.

Abstract: Fructosamine values in two groups of hypo- and hyperthyroid patients were compared with the values in a reference group of non-diabetics. In hyperthyroid patients the fructosamine values were significantly lower than in the reference group. Also the mean concentrations of albumin and total protein in serum are significantly lower for hyperthyroid patients compared to hypothyroid patients. The results do not provide evidence for a simple relationship between fructosamine and protein values in these patient groups. Therefore we do not recommend to relate fructosamine to protein or albumin using correction factors. Under conditions of thyrotoxicosis fructosamine is no reliable indicator of previous serum glucose concentrations. The test is not affected by monoclonal IgG gammopathy.

Abstract: Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isoenzyme-3 (LD-3) has been isolated in milligram quantities from human erythrocytes. Using an improved procedure--which involves complete hemolysis of the erythrocytes, diethylaminoethyl (DEAE)-Sephacel column chromatography, and 5'-AMP-Sepharose 4B affinity chromatography--we obtained 23,000-fold purified isoenzyme from the crude hemolysate (overall yield about 90%). The final product was homogeneous on polyacrylamide disc gel electrophoresis and had a specific activity of about 435 kU/g. Its amino acid composition is presented. With the eventual aim to make visible and isolate IgA kappa antibody-secreting B lymphocytes, we developed reproducible methods for preparing fluorescein isothiocyanate isomer-1-conjugated LD-3 with a fluorescein/LD-3 molar ratio between 1.3 and 3.3, and biotinylated LD-3 with a biotin/LD-3 molar ratio between 1.3 and 2.5. In evaluating the stability of these two conjugates, we determined that they still can react with IgA kappa to form the IgA kappa (LD-3)2 complex.

Abstract: We have purified with a cumulative recovery of 48% from the serum of a patient the immunoglobulin A kappa subunit of the lactate dehydrogenase-immunoglobulin A kappa (LD-IgA kappa) complex. It appears that the pI range of the complex is 5.4-5.8. The Ig part of the complex showed a monoclonal character, and the complex exhibited a 1:2 molar ratio of the Ig to the LD isoenzyme. From reconstitution experiments by two different methods we concluded that LD-3 is essential for restoring the original complex. Additional studies showed a recombination of the IgA kappa with both autologous and homologous LD-3, and a binding of LD-3 at the Fab region of the Ig. The estimated value of the affinity constant (Keq) was 2.1 X 10(9) liters/mol. Analysis of the specific LD-3-binding IgA kappa concentrations in the sera of five cases revealed a broad range of the individual immune response. Our first quantitative data on the lymphocyte subpopulations revealed a significantly increased OKT4/OKT8 ratio due to a reduction in the absolute number of T suppressor cells.

Abstract: The existence of a lactate dehydrogenase-immunoglobulin A kappa complex was demonstrated as a marked increase of lactate dehydrogenase activity at the lactate dehydrogenase-3 band in the agar-agarose gel-electrophoresis pattern of sera from seven patients from an unselected group of 21 800 patients. The complex was isolated, in an almost pure form, by gel filtration and affinity chromatography, from the serum of a patient with circulating hepatitis B surface antigen. The complex had a relative molecular mass (Mr) of approximately 445 000 as determined by gel filtration. Electrophoresis in sodium dodecyl sulfate in the absence of reducing agents showed the presence of two subunit bands with Mr 170 000 and 34 000. We therefore propose that the native complex consists of one monomeric immunoglobulin A kappa linked to two tetrameric lactate dehydrogenase molecules. The enzymic activity of the complex is probably from lactate dehydrogenase isoenzyme-5.

Abstract: The existence of an alkaline phosphatase-lipoprotein-X complex was demonstrated as an abnormally moving band in the agar-agarose gel alkaline phosphatase pattern of sera from 58 patients in a group of 72 patients whose sera contained lipoprotein-X. The position of the abnormally moving band in the agar-agarose gel alkaline phosphatase pattern and the percentage of alkaline phosphatase activity in the complex in relation to the total serum alkaline phosphatase activity depend on the serum lipoprotein-X concentration. The presence of the complex is a reliable indicator for cholestasis and of limited value in the differentiation between intrahepatic and extrahepatic cholestasis.

Abstract: An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.

Abstract: Four cases of newborn infants whose mothers had a low urinary oestrogen excretion during pregnancy are reported. Placental sulphatase deficiency in placental insufficiency were excluded by a dehydroepiandrosterone sulphate loading test. Postnatally, they developed a clinical picture characterized by an inappropriate secretion of cortisol which, by the results of an adrenocorticotrophic hormone stimulation test, appeared to be due to an impairment of adrenocorticotrophic hormone secretion. The prenatal and postnatal steroid metabolism is discussed.

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Abstract: After the introduction, which provides a cursory overview of the biochemical role of lactate dehydrogenase (LD) as well as a short historical survey of this enzyme and its complexes, a successful attempt to isolate the immunoglobulin (Ig) component of the LD-IgA[kappa] complex is described. Recombination experiments between purified IgA[kappa] and purified LD-isoenzymes identified LD-3 as the counterpart of the complex. Moreover, our experimental results suggest that the occurrence of the IgA[kappa] is not due to molecular alterations of the LD-3. Of five subjects, the serum concentration of complexed cross-reacting IgA[kappa] was determined. The data show a wide range of cross-reacting IgA[kappa] within this study population. On the basis of the consistent features between the (LD-3)2-IgA[kappa] complex and macro creatine kinase 1, we propose to term the complex: macro LD type 1.

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Abstract: Six peptides have been isolated from a tryptic digest of rabbit plasma by ultrafiltration, gel filtration and ion-exchange chromatography: they have been characterized by their isoelectric points, amino acid composition, N-terminal amino acids and biological activities. Two of these peptides potentiated bradykinin, one (peptide A-VI) by a factor of 2 at a concentration of 0.09 mM and the other (peptide A-VII) by a factor of 2 at a concentration of 0.07 mM. Analysis by phenylisothiocyanate degradation as well as by mass spectrometry revealed that the amino acid sequence of peptide A-VI was: Leu-Val-Glu-Ser-Ser-Lys, while that of peptide A-VII was: Thr-Pro-Val-Ser-Glu-Lys. These biologically active peptides were found to originate from the albumin. The amino acid sequence of a biologically inactive peptide (A-VIII) as determined by mass spectrometry was: Ala-(Ile,Leu)-Ser-Ala-Ala-Gln-Glu-Arg. Ten peptides have been isolated from a tryptic digest of bovine albumin. Two of these potentiated bradykinin, one (peptide C-IV) by a factor of 2 at a concentration of 0.07 mM and the other (peptide D-V) by a factor of 2 at a concentration of 0.13 mM. Peptide C-IV had the same sequence as peptide A-VII, while the amino acid sequence of peptide D-V was: Ile-Glu-Thr-Met-Arg. The following peptides have been synthesized by the solid phase method and purified by ion-exchange chromatography: Leu-Val-Glu-Ser-Ser-Lys (peptide A-VI), Val-Glu-Ser-Ser-Lys and Val-Glu-Ser-Lys. The first one potentiated bradykinin by a factor of 2 at a concentration of 0.11 mM, the second one by a factor of 2 at a concentration of 0.13 mM, while the third one was biologically inactive at a concentration of 1.7 mM. A possible relationship between structure and function of these peptides is discussed.