Strategy for determining racial and environmental similarities and differences for plasma proteins.

The aim of this protocol is to establish a common basis for the production of reference values and well-defined and documented reference intervals for plasma proteins, based on common standardization, using the IFCC/BCR/CAP Certified Reference Material CRM 470. The strategy is to search for racial and environmental/geographical similarities and sources of differences in order to describe the main causes for variability among smaller or larger groups in selected societies and to estimate the sizes of differences for the different proteins according to the investigated sources. For this purpose, groups of reference individuals are selected according to race and geographical/environmental location, e.g. African Americans and Caucasians from the US. The reference individuals are groups of approximately 160 healthy male blood donors, 20 to 60 years of age. Rule-out criteria are positivity for HIV, hepatitis B and C antibodies and blood hemoglobin below the lower reference limit. Exclusion in relation to different C-reactive protein (CRP) levels will be investigated. Coagulation, storage conditions, transport, and the procedure for thawing are specified. The laboratories undertaking the measurements must have adequate analytical performance, and calibration and quality of performance are defined and documented, together with recommended control materials and procedures. Statistical models for describing distributions and for comparing groups are described. It is recommended that the data be presented as reference limits with 90% confidence intervals of those limits.

Effect of a new international reference preparation for proteins in human serum (certified reference material 470) on results of the College of American Pathologists Surveys for plasma proteins.

OBJECTIVE: To examine the impact of a new international reference preparation for serum proteins on the among-manufacturer variance in the College of American Pathologists Surveys. DATA SOURCE: The results of the Surveys for the years 1993-1998, supplied by the College of American Pathologists. DATA EXTRACTION AND SYNTHESIS: Mean values for manufacturers' assays were compared for each protein in the quality control samples. Results were separated by the reported reference material from which values for calibrators had been transferred. Standard statistical parameters (means, standard deviations, and coefficients of variation) were calculated from the reported means. CONCLUSIONS: Among-manufacturer coefficients of variation have dropped significantly for most serum proteins since the introduction of the new reference material. Possible reasons for continued differences are discussed.

Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges for 14 proteins in serum based on the standardization against the IFCC/BCR/CAP Reference Material (CRM 470). International Federation of Clinical Chemistry. Community Bureau of Reference of the Commission of the European Communities. College of American Pathologists.

The release in 1993 of a new reference material for serum proteins, CRM 470/RPPHS 5 has given rise to a great improvement in the between-laboratory variability of serum protein measurements worldwide. Conversion to the new reference material results in significant changes in reference values for some proteins. The establishment of new reference ranges will take a considerable time, and in the interim several professional societies and diagnostic companies have agreed to use consensus reference ranges based on studies already undertaken.

Protein binding to the low density lipoprotein receptor promoter in vivo is differentially affected by gene activation in primary human cells.

Protein-DNA interactions within a region of the LDL receptor promoter involved in sterol-mediated feedback repression of transcription were examined using in vivo genomic footprinting with dimethylsulfate (DMS). A broad region of protein-DNA contacts spanning from repeat 1 to beyond the transcription start sites was observed in primary cultures of human skin fibroblasts and hepatocytes. Hypermethylation of guanine -59 within the sterol regulatory element-1 (SRE-1, repeat 2) occurred within a 4.0 h incubation of fibroblasts with media containing lipoprotein-deficient serum (LPDS) and cholesterol synthesis inhibitors. Methylation of this residue was reduced to control levels within 2.0 h after the addition of a mixture of 25-hydroxycholesterol and mevalonic acid. The time-dependent changes in DMS-reactivity of guanine -59 induced by the cholesterol synthesis inhibitors or oxysterols were paralleled by alterations in LDL receptor mRNA. In contrast to the results with fibroblasts, neither cholesterol synthesis inhibitors nor oxysterols produced consistent effects on the DMS-reactivity of guanine -59 in hepatocytes despite induction or repression of LDL receptor mRNA in these cells. Interestingly, no other changes in the protection pattern over repeats 1, 2, and 3 were apparent in either fibroblasts or hepatocytes. These results demonstrate that hypermethylation of guainine -59 within the SRE-1 is positively associated with activation of LDL receptor gene transcription in skin fibroblasts. Furthermore, the absence of demonstrable changes in DMS-reactivity of other purines within this region suggests that the LDL receptor promoter is poised to activate transcription with only minimal changes of protein binding to the proximal promoter in vivo.

Serum levels of androgens and estrogens and 'steroid-sensitive' liver proteins in early human pregnancy: influence on the gender of the offspring.

Serum androgens, estrogens, 'steroid-sensitive proteins', thyroid components, and albumin were measured twice within a 4-5 week interval in 44 cases of early normal pregnancy (gestational weeks 8-18). Positive correlations were found in the total material between dehydroepiandrosterone sulfate (DHAS) and testosterone (T), unconjugated and total estrone, albumin, tetraiodothyronine, and calculated free tetraiodothyronine concentrations and within 2-week intervals between DHAS and T, estradiol-17 beta, and unconjugated and total estrone, and between T and estradiol-17 beta and unconjugated estrone. Positive correlations were also found between the rates of change in DHAS of albumin. No significant association was found between 'steroid-sensitive proteins' and androgens or androgen/estrogen ratios. Women giving birth to girls had significantly higher serum levels of pregnancy-associated alpha 2-macroglobulin, thyroxine-binding globulin, and ceruloplasmin and lower ratios between T and sex hormone binding globulin than women giving birth to boys. The important role of placental aromatization in the metabolism of maternal androgens is well known. Albumin binding and thyroid status may also affect the metabolism of DHAS during pregnancy. The androgen/estrogen balance may be of little importance for the regulation of 'steroid-sensitive' proteins during early pregnancy. The mechanism behind the higher serum levels of pregnancy-associated alpha 2-macroglobulin, thyroxine-binding globulin, and ceruloplasmin in women giving birth to girls remains to be elucidated.

Diagnostic and prognostic value of interleukin-6 and C-reactive protein in community-acquired pneumonia.

The diagnostic and prognostic value of admission serum levels of interleukin-6 (IL-6) and C-reactive protein (CRP) was investigated in 203 hospital-treated patients with community-acquired pneumonia (CAP). In serum samples obtained during the first 24 h after admission, IL-6 was detectable in 198 patients (98%), with a median value of 50 ng/l. Ten % of the patients had IL-6 values of 1000 ng/l. A clear positive correlation between IL-6 and CRP was found (r = 0.29, p < 0.0001). Patients with high IL-6 or CRP levels had longer duration of fever, longer hospital stay, and had less often recovered clinically or radiographically on follow-up weeks after discharge. A high IL-6, but not a high CRP, also seemed to be associated with a higher mortality. Bacteremic pneumococcal pneumonia had the highest levels of IL-6 (mean 2852 and median 420 ng/l) and CRP (mean 292 and median 285 mg/l). High IL-6 values were also seen in patients with non-bacteremic pneumococcal pneumonia, while all patients with pneumonia due to other bacterial, or viral, aetiology had IL-6 levels of < or = 300 ng/l. In conclusion, IL-6 and CRP are promising diagnostic and prognostic tools in the management of CAP. Further studies are needed to establish the usefulness of repeated measurements early in the hospital course of the disease.

The diagnostic value of repetitive preoperative analyses of C-reactive protein and total leucocyte count in patients with suspected acute appendicitis.

BACKGROUND: Studies have shown that C-reactive protein (CRP) and total leucocyte count (WBC) in suspected acute appendicitis analysed only on admission gave valuable information to guide the surgeon. The aim of this study was to investigate the usefulness of CRP and WBC measured repetitively before operation. METHODS: During a 1-year period 227 patients were studied before emergency appendicectomy. CRP and WBC were analysed every 4th h. The upper limits of the reference intervals used were 9.0 x 10(9)/l for WBC and 10 mg/l for CRP. RESULTS: Of the 227 appendicectomized patients, 170 (75%) had acute appendicitis. Sixty-six of the patients were tested on two or more occasions every 4th h. Forty-six of these patients had appendicitis; repetitive tests showed a continuing rise in CRP values but a continuing decrease in WBC. The negative appendicectomy rate among these 66 patients was 30%, which theoretically would have fallen to 19% if patients with normal results had not been subjected to surgery. CONCLUSIONS: Repeated laboratory tests for CRP and WBC should be performed in patients with suspected acute appendicitis requested to stay for further observation. If these test results are normal, the surgeon should preferably refrain from operating but consider other differential diagnoses.

New international reference preparation for proteins in human serum (RPPHS).

Quality-control surveys in recent years, in various parts of the world, have shown poor between-laboratory agreement for measurements of plasma proteins. Despite the existence of international reference materials distributed by the World Health Organization, standards produced by diagnostics manufacturers and professional organizations differ significantly in their ascribed values. The reasons for this are complex but include poor availability of the primary materials, confusion about their use, and the fact that their turbidity on reconstitution precludes their use in modern optical immunoassays. This unfortunate situation led to an important initiative to produce sufficient quantities of a widely available, optically clear secondary reference material for plasma proteins that could be used worldwide by manufacturers, professional organizations, and laboratories. Here we present an overview on how the laboratory community, including manufacturers, clinical laboratories, professional societies, and regulators, has reached what we consider is a successful conclusion to a difficult problem.

Ketoconazole and 25-hydroxycholesterol produce reciprocal changes in the rate of transcription of the human LDL receptor gene.

Sterol-dependent regulation of low-density lipoprotein (LDL) receptor gene expression was studied in the human hepatoma HepG2 cell line. Incubation of HepG2 cells with 20 microM ketoconazole increased the level of LDL receptor mRNA. After a lag of approx. 1.0 h the level rose 6.5-fold within 8.0 h and remained elevated for up to 24 h. Incubation with 10 micrograms 25-hydroxycholesterol/ml for 24 h produced a 40-50% reduction in the level of LDL receptor mRNA. Ketoconazole- and 25-hydroxycholesterol-induced changes in LDL receptor mRNA accumulation were due to alterations in the relative rate of LDL receptor gene transcription as measured by nuclear run-on transcription. Incubation with 20 microM ketoconazole for 4 h or 10 micrograms 25-hydroxycholesterol/ml for 24 h produced a 3.6-fold increase and a 40% reduction, respectively, in the transcription rate of LDL receptor gene. Removal of the Alu-like sequence elements within the LDL receptor cDNA was required to consistently measure changes in LDL receptor gene transcription. No significant changes were noted in the half-life of LDL receptor mRNA in ketoconazole or 25-hydroxycholesterol-treated cells. These data demonstrate that sterol-dependent changes in the level of LDL receptor mRNA can be completely accounted for by changes in the rate of LDL receptor gene transcription.

Transthyretin messenger ribonucleic acid expression in the pancreas and in endocrine tumors of the pancreas and gut.

Transthyretin (TTR) cDNA probes were used to determine the presence of TTR mRNA in Northern blots from rat, porcine, and human organs as well as from human endocrine tumors. We also used in vitro translation in our study of the human tissues. In accordance with previous findings, TTR mRNA was found in the choroid plexus and, to a lesser extent, in the liver of all three species. In addition low levels of TTR mRNA were identified in the rat and human pancreas. All of the endocrine pancreatic tumors (two glucagonomas, two insulinomas, and one nonfunctional tumor) and the gut carcinoid also contained TTR mRNA, whereas other endocrine tumors (two pheochromocytomas and one paraganglioma) and one adenocarcinoma of the pancreas were TTR mRNA negative. The level of TTR mRNA expression in most of the endocrine pancreatic tumors exceeded that in the liver. The in vitro translations produced pre-TTR molecules of similar size for all TTR mRNA-positive human organs and tumors.

Transthyretin in endocrine pancreatic tumors.

The occurrence of transthyretin (TTR) in 25 endocrine pancreatic tumors was investigated by immunohistochemical methods using both polyclonal and monoclonal antibodies. All malignant insulinomas were strongly TTR immunoreactive, more so than their benign counterparts, which in some cases were TTR negative. All glucagonomas and nonfunctioning tumors were TTR immunoreactive, whereas gastrinomas and VIPomas were TTR negative. TTR, chromogranin A, and the argyrophil reaction (Grimelius' silver technique) had similar distributions among the cells in many, but not all, tumors. Coexistence of TTR with glucagon, insulin, or pancreatic polypeptide in tumor cells was demonstrated. TTR was also quantitated in preoperative serum samples by electroimmuno assay in some cases. Although one patient with a glucagonoma had a markedly increased serum TTR level, five other patients with endocrine tumors, including two patients with glucagonoma, had TTR levels in serum that were within or below the reference range.

Transthyretin microheterogeneity and thyroxine binding are influenced by non-amino acid components and glutathione constituents.

Two non-amino acid components as well as the glutathione constituents in labile associations with transthyretin (TTR) have been detected by preparative polyacrylamide gel electrophoresis from preparations isolated by affinity chromatography on Sepharose-bound retinol-binding protein (RBP). Incubation of native or reduced TTR with these novel components influenced the quaternary structure and caused reactions with reduced TTR in particular. Reduction of isolated TTR monomers released cysteine from the quantitatively major monomer, but non-amino-acid components from another dominating monomer. The reaction patterns also influence thyroxine (T4) binding. These relationships indicate that interactions in serum of TTR with constituents of glutathione and components different from T4 and retinol-RBP are important for the metabolism and function of TTR.

Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets.

We examined transthyretin immunoreactivity (TTR-IR) in human and porcine liver, choroid plexus, and pancreatic islets with both polyclonal and monoclonal antibodies to TTR. The specificity of the immunoreactions and the effects of various fixatives were tested in immunohistochemical and dot-blot systems. B-5 fixative (mercuric chloride and sodium acetate in formalin) was the best immunopreservative. In both species, the TTR-IR in choroid plexus epithelial cells was strong and was much greater than that in hepatocytes. Glucagon cells in pancreatic islets were also strongly TTR immunoreactive. Insulin cells were slightly TTR immunoreactive in human but strongly so in porcine pancreas. The finding of TTR-IR in normal islets explains the presence of TTR-IR in human endocrine pancreatic tumors, notably glucagonomas and malignant insulinomas.

Serum levels of estrogens and of five 'steroid sensitive' proteins in early normal pregnancy.

Serum concentrations of sex hormone binding globulin (SHBG), pregnancy associated alpha 2-glycoprotein (alpha 2-PAG), caeruloplasmin (Cp), alpha 1-antitrypsin (alpha 1-At) and transferrin (Tf), unconjugated and total estrone, and unconjugated estradiol-17 beta were measured twice at a 4-5 week interval in 43 cases of early normal pregnancy (gestational weeks 6-19). Significant correlations between estrogen and protein levels in the total material were observed for all proteins except for Tf. However, within 2-week intervals of pregnancy, significant correlations were obtained only in certain intervals between estrogens on one hand and SHBG, Cp and alpha 1-At on the other. Significant correlations between rates of increase of estrogens and of proteins were obtained only at certain occasions for SHBG, Cp, Tf and alpha 1-At. The results indicate that, although estrogens may have a modulatory function in 'steroid-sensitive' protein synthesis, they are probably not the main physiological regulator.

Different types of microheterogeneity of human thyroxine-binding prealbumin.

Human thyroxine-binding prealbumin or transthyretin has been isolated by three different methods. In all cases, the isolated material is microheterogeneous in an isoelectric focusing system, revealing a pattern of at least 10 bands. These subforms represent tetramers. Dissociation of the protein in serum samples and dissociation in the presence of urea or sodium dodecyl sulfate (SDS) reveal eight other forms, differing in isoelectric point and concluded to be monomers. Two different sets of dissociated forms are identified, one from urea treatment and the other from SDS treatment. The latter set is apparently also present in serum. Interpretations are complicated by multiplicities of all forms, atypical electrophoretic migrations, nonidentical effects of urea and SDS, and the absence of dimer under most conditions. However, reassociations of monomers and formation of interspecies hybrid molecules identify dimers and clarify interpretations of the dissociated sets. In at least five of the forms likely to represent monomers, the difference is dependent on the nature of the SH group at Cys-10, which can be oxidized, can be reduced, or can be in a mixed disulfide, probably with glutathione and its degradation products. Amino acid sequence analysis reveals an additional N-terminal heterogeneity (with start at positions 1, 2, and 3) of the three most abundant monomers, but this does not explain the observed differences among monomeric forms. It is concluded that prealbumin exhibits different types of microheterogeneity, the major pattern of which is explained by the status of the subunit SH group and the ability of prealbumin to dissociate into monomers. A relation appears to exist between SH group status on the one hand, and retinol-binding protein interaction, the ability to dissociate into monomers, and possibly also binding of thyroxine on the other. These interactions suggest complex functional relationships for prealbumin in serum.

Clinical manifestations of Borrelia infections of the nervous system.

Clinical, treatment and laboratory parameters were analyzed in 46 consecutive Swedish patients with Borrelia infections of the nervous system. The importance of age in the clinical symptoms, the wide spectrum of disease, and the chronic behaviour of the Borrelia infection of the nervous system was stressed, as well as the benefit of high-dose intravenous antibiotics, especially penicillin G. Borrelia infection of the nervous system can imitate other diseases. When associated with meningitis it can mimic psychosomatic disorders, when associated with radiculoneuritis it may imitate herniated discs and when central nervous involvement of the Borrelia infection occurs, it can mimic a non-infectious, thrombotic or haemorrhagic cerebro-vascular disease.

Oestrogen and thyroxine-binding globulin levels in early normal pregnancy.

Peripheral serum levels of thyroxine-binding globulin (TBG), unconjugated and total oestrone, and unconjugated oestradiol-17 beta were measured twice in 60 cases of early normal pregnancy (gestational weeks 6-19). Although highly significant correlations between TBG and oestrogen levels were obtained in the total material, the correlations within two-week intervals of pregnancy were rather poor and mostly insignificant. Furthermore, there were no associations at all between the increase rates of TBG and those of the oestrogens, either within two-week intervals or in the total material. The findings do not support the common concept of continuously rising oestrogen stimulation as the main factor affecting the TBG synthesis during pregnancy.

Monoclonal antibodies to transthyretin.

Monoclonal antibodies (mabs) have been raised against human transthyretin (hTTR). The protein was isolated by an affinity chromatography procedure using Sepharose-hRBP and BALB/c mice were immunized. Following fusion with SP 2/0 myeloma cells, 26 single cell clones producing antibodies against hTTR were isolated. These mabs have been characterized and efforts have been made to establish the epitopes that they recognize. So far at least two different epitopes have been identified both residing in a mid-region fragment corresponding to the amino acid sequence 35-103 of the hTTR subunit. All mabs have been found suitable for immunohistochemical localization of hTTR even in formaldehyde fixed and paraffin embedded tissues.

Influence of blood group on the availability of receptors for attachment of uropathogenic Escherichia coli.

Escherichia coli strains with defined receptor specificity were used as probes to analyze the individual variation in host cell receptors with respect to blood groups. The adhesins were initially characterized as mannose sensitive (MS), mannose resistant (MR), or nonagglutinating (-). The receptor specificity of the strains with MR adhesins was defined by agglutination of synthetic Gal alpha 1----4Gal beta covalently linked via a spacer arm, (CH2)2S(CH2)2CO approximately H-bovine serum albumin (BSA) to BSA-latex beads as specific for the globoseries glycolipid receptors (MR:GS). Strains with MR adhesins not reacting with Gal alpha 1----4Gal beta-BSA-latex were designated MR:nonGS. The attachment and hemagglutination of the MR:GS strains was strictly dependent on Gal alpha 1----4Gal beta-containing receptors, as shown by the absence of binding to cells from individuals of blood group P lacking these structures. Previous reports showed differences in the composition of globoseries glycolipids between erythrocytes from individuals of P1 and P2. No significant difference was found, However, in the mean adhesion to P1 and P2 epithelial cells or in the agglutination titer for P1 and P2 erythrocytes. The MR:GS receptors were equally distributed on squamous and transitional epithelial cells. In contrast, the distribution of MR:nonGS receptors was skewed. Attachment occurred mostly to squamous epithelial cells. The attachment of strains with MR:nonGS adhesins was independent of the P blood group of the cell donor. The binding ability of MR:GS and MR:nonGS adhesins appeared independent and additive. The attachment was not influenced by the ABH blood group. However, increased binding to epithelial cells from nonsecretors occurred regardless of the P blood group, suggesting a shielding of receptors by products controlled by the secretor genes. These results illustrate how individual variation in cell surface components with and without receptor activity determine the interaction of a ligand with a known receptor.

Thyroxine-binding globulin in spontaneous abortion.

Thyroxine-binding globulin (TBG), thyroxine-binding prealbumin (TBPA), thyroxine (T4), free T4, triiodothyronine (T3) and thyroid-stimulating hormone (TSH) were studied in 144 apparently healthy, euthyroid pregnant women displaying miscarriages. In an additional group of 18 aborting women a frozen blood sample drawn in early pregnancy was similarly analyzed for the thyroid components as well as for human chorionic gonadotropin (HCG). The values were compared to those of normal pregnancies (n = 228) at corresponding intervals. Significantly lower values of TBG were found in the spontaneously aborting group. A significantly lower value of TBG was found to be more predictive concerning spontaneous abortion than that of HCG.