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Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Carbohydrate-deficient transferrin (CDT) is used as a biomarker of sustained high alcohol consumption. The currently available measurement procedures for CDT are based on various analytical techniques (HPLC, capillary electrophoresis, nephelometry), some differing in the definition of the analyte and using different reference intervals and cut-off values. The Working Group on Standardization of CDT (WG-CDT), initiated by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), has validated an HPLC candidate reference measurement procedure (cRMP) for CDT (% disialotransferrin to total transferrin based on peak areas), demonstrating that it is suitable as a reference measurement procedure (RMP) for CDT. Presented is a detailed description of the cRMP and its calibration. Practical aspects on how to treat genetic variant and so-called di-tri bridge samples are described. Results of method performance characteristics, as demanded by ISO 15189 and ISO 15193, are given, as well as the reference interval and measurement uncertainty and how to deal with that in routine use. The correlation of the cRMP with commercial CDT procedures and the performance of the cRMP in a network of laboratories are also presented. The performance of the CDT cRMP in combination with previously developed commutable calibrators allows for standardization of the currently available commercial measurement procedures for CDT. The cRMP has recently been approved by the IFCC and will be from now on be known as the IFCC-RMP for CDT, while CDT results standardized according to this RMP should be indicated as CDT IFCC ., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The need for equivalent results of routine measurement procedures for the alcohol biomarker carbohydrate-deficient transferrin (CDT) has been recognized by the IFCC. This article describes a project to harmonize CDT as conducted by an IFCC working group initiated for this purpose., Methods: We used procedures for achieving harmonization as developed by the Consortium for Harmonization of Clinical Laboratory Results to assess the suitability of a candidate reference measurement procedure (cRMP), candidate reference materials (cRMs), and the success of efforts to achieve harmonization., Results: CDT measurement procedures in routine use showed good reproducibility (CV 1.1%-2.8%) and linearity (r > 0.990) with variable slopes (0.766-1.065) and intercepts (-0.34 to 0.92) compared to the cRMP. Heterogeneity after simulated harmonization was 4.7%. cRMs of frozen human native sera demonstrated commutability and 3-year stability for routine measurement procedures. The cRMP provided reproducible value assignment to cRMs with an expanded uncertainty (k = 2) of 0.03% at the 1.2% CDT level and 0.06% at the 4.4% CDT level. Harmonization efforts reduced the intermeasurement CV from 8.8% to 3.4%, allowed 99% recovery of the values assigned with the cRMP, and demonstrated 99% of results within the desirable allowable total error. Harmonization was less successful in samples with low CDT and high trisialotransferrin concentrations., Conclusions: Harmonization of CDT is possible with frozen human native sera as cRMs with values assigned by use of the cRMP. We propose the cRMP as a candidate international conventional reference measurement procedure and cRMs as candidate international calibrators., (© 2014 The American Association for Clinical Chemistry.)

Carbohydrate-deficient transferrin (CDT) is a generic term that refers to the transferrin glycoforms whose concentration in blood is temporarily increased by sustained alcohol consumption. Due to high clinical specificity, CDT was proposed as a biomarker of heavy alcohol use and has been available for about 20 years. A number of methods have been developed for CDT measurement based on different analytical techniques and principles and without any harmonization or calibration to a reference method. As a consequence, neither the reference limits nor the cut-off values have been similar across assays, hampering understanding of the diagnostic value of CDT and its routine use. This prompted the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to initiate a Working Group on Standardization of CDT (WG-CDT). This third publication of the WG-CDT is devoted to testing the commutability of native and disialotransferrin-spiked serum panels as candidate secondary reference materials, in order to prove the harmonization potential of commercial CDT methods. The results showed that assay harmonization reduced the inter-laboratory imprecision in a network of reference laboratories running the HPLC candidate reference method. In the seven commercial methods evaluated in this study, the use of multi-level secondary calibrators of human serum origin significantly reduced the between-method imprecision. Thus, harmonization of CDT measurements by different methods can be achieved using this calibration system, opening the way for a full standardization of commercial methods against a reference method by use of certified reference materials.

Carbohydrate-deficient transferrin (CDT) is a descriptive term used for a temporary change in the transferrin glycosylation profile caused by alcohol, and used as a biomarker of chronic high alcohol consumption. The use of an array of methods for measurement of CDT in various absolute or relative amounts, and sometimes covering different transferrin glycoforms, has complicated the comparability of results and caused confusion among medical staff. This situation prompted initiation of an IFCC Working Group on CDT standardization. This second publication of the WG-CDT covers the establishment of a network of reference laboratories running a high-performance liquid chromatography (HPLC) candidate reference measurement procedure, and evaluation of candidate secondary reference materials. The network laboratories demonstrated good and reproducible performance and thus can be used to assign target values for calibrators and controls. A candidate secondary reference material based on native human serum lyophilized with a cryo-/lyoprotectant to prevent protein denaturation was found to be commutable and stable during storage. A proposed strategy for calibration of different CDT methods is also presented. In an external quality assurance study involving 66 laboratories and covering the current routine CDT assays (HPLC, capillary electrophoresis and immunoassay), recalculation of observed results based on the nominal values for the candidate calibrator reduced the overall coefficient of variation from 18.9% to 5.5%. The logistics for distribution of reference materials and review of results were found to be functional, indicating that a full reference system for CDT may soon be available.

Background: The American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD)/International Diabetes Federation (IDF)/IFCC Consensus Statement on the worldwide standardization of HbA(1c) states that "... [HbA(1c)] results are to be reported world-wide in IFCC units ... and derived NGSP units ... , using the IFCC-NGSP master equation.", Methods: We describe statistical methods to evaluate and monitor the relationships as expressed in master equations (MEs) between the IFCC Reference Measurement procedure (IFCC-RM) and designated comparison methods (DCMs) [US National Glycohemoglobin Standardization Program (NGSP), Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC), and Mono-S in Sweden]. We applied these statistics, including uncertainty calculations, to 12 studies in which networks of reference laboratories participated, operating the IFCC-RM and DCMs., Results: For NGSP and Mono-S, slope, intercept, and derived percentage HbA(1c) at the therapeutic target show compliance with the respective MEs in all 12 studies. For JDS/JSCC, a slight deviation is seen in slope and derived percentage HbA(1c) in 2 of the 12 studies. Using the MEs, the uncertainty in an assigned value increases from 0.42 mmol/mol HbA(1c) (IFCC-RM) to 0.47 (NGSP), 0.49 (JDS/JSCC), and 0.51 (Mono-S)., Conclusions: We describe sound statistical methods for the investigation of relations between networks of reference laboratories. Application of these statistical methods to the relationship between the IFCC-RM and DCMs in the US, Japan, and Sweden shows that they are suitable for the purpose, and the results support the applicability of the ADA/EASD/IDF/IFCC Consensus Statement on HbA1c measurement.

Background: The IFCC Reference Measurement System for hemoglobin (Hb)A(1c) (IFCC-RM) has been developed within the framework of metrologic traceability and is embedded in a network of 14 reference laboratories. This paper describes the outcome of 12 intercomparison studies (periodic evaluations to control essential elements of the IFCC-RM)., Methods: Each study included: unknown samples (to test individual network laboratories); known samples (controls); recently manufactured calibrators (to check calculated assigned value); stored calibrators (to test stability) and a calibration-set (to calibrate the IFCC-RM). The unknown samples are measured by use of the IFCC-RM and the designated comparison methods [DCMs; the National Glycohemoglobin Standardization Program (NGSP) in the US, Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC) in Japan, and Mono-S in Sweden] are used to investigate the stability of the Master Equation (ME), the relationship between IFCC-RM and DCMs., Results: A total of 105 IFCC-RM data sets were evaluated: 95 were approved, 5 were not, and for 5 no data were submitted. Trend analysis of the MEs, expressed as change in percentage HbA(1c) per year, revealed 0.000% (NGSP, not significant), -0.030%, (JDS/JSCC; significant) and -0.016% (Mono-S; not significant). Evaluation of long-term performance revealed no systematic change over time; 2 laboratories showed significant bias, 1 poor reproducibility. The mean HbA(1c) determined by laboratories performing mass spectrometry (MS) was the same as the mean determined by laboratories using capillary electrophoresis (CE), but the reproducibility at laboratories using CE was better. One batch of new calibrators was not approved. All stored calibrators were stable., Conclusion: A sound reference system is in place to ensure continuity and stability of the analytical anchor for HbA(1c).

The measurement of glycated hemoglobin is central in the monitoring of glycemic control in patients with diabetes. There are at least 30 different laboratory assays commercially available to measure the proportion of HbA1c in blood. In 1995 the IFCC established a Working Group (IFCC WG-HbA1c) to achieve international standardization of HbA1c measurement. The main achievements can be summarized as follows: a) a reference measurement procedure has been established with purified primary calibrators; b) a network of reference laboratories has been developed worldwide; and c) work has begun on implementation of traceability to the IFCC reference system. The IFCC WG-HbA1c recognizes the recommendation of the IFCC-IUPAC Committee on Nomenclature, Properties and Units that the analyte measured by the IFCC reference measurement procedure has been defined as betaN1-deoxyfructosyl-hemoglobin and that the recommended measurement units are mmol/mol. The IFCC WG-HbA1c recommends maintaining the use of the name HbA1c in clinical practice.

An alcohol-associated change in the serum transferrin glycoform pattern, carbohydrate-deficient transferrin (CDT), is used as a biomarker of chronic moderate to heavy alcohol consumption. A current limitation in CDT analysis is the lack of standardization, which hampers clinical and analytical comparison between studies. This situation prompted initiation of a Working Group (WG) on CDT Standardization under the auspices of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). The standardization work aims to define and validate the analyte, select a reference method, work out procedures for the production of reference materials, and make suggestions for the clinical usage of CDT. The first recommendation of the WG is that disialotransferrin should be the primary target molecule for CDT measurement and the single analyte on which CDT standardization is based. It is further recommended that HPLC should be the analytical principle considered as the basis of an interim reference method until a suitable mass spectrometric reference method is established. In clinical use, CDT should be expressed in a relative amount (% CDT), to compensate for variations in the total transferrin concentration.

Background: The national programs for the harmonization of hemoglobin (Hb)A(1c) measurements in the US [National Glycohemoglobin Standardization Program (NGSP)], Japan [Japanese Diabetes Society (JDS)/Japanese Society of Clinical Chemistry (JSCC)], and Sweden are based on different designated comparison methods (DCMs). The future basis for international standardization will be the reference system developed by the IFCC Working Group on HbA(1c) Standardization. The aim of the present study was to determine the relationships between the IFCC Reference Method (RM) and the DCMs., Methods: Four method-comparison studies were performed in 2001-2003. In each study five to eight pooled blood samples were measured by 11 reference laboratories of the IFCC Network of Reference Laboratories, 9 Secondary Reference Laboratories of the NGSP, 3 reference laboratories of the JDS/JSCC program, and a Swedish reference laboratory. Regression equations were determined for the relationship between the IFCC RM and each of the DCMs., Results: Significant differences were observed between the HbA(1c) results of the IFCC RM and those of the DCMs. Significant differences were also demonstrated between the three DCMs. However, in all cases the relationship of the DCMs with the RM were linear. There were no statistically significant differences between the regression equations calculated for each of the four studies; therefore, the results could be combined. The relationship is described by the following regression equations: NGSP-HbA(1c) = 0.915(IFCC-HbA(1c)) + 2.15% (r(2) = 0.998); JDS/JSCC-HbA(1c) = 0.927(IFCC-HbA(1c)) + 1.73% (r(2) = 0.997); Swedish-HbA(1c) = 0.989(IFCC-HbA(1c)) + 0.88% (r(2) = 0.996)., Conclusion: There is a firm and reproducible link between the IFCC RM and DCM HbA(1c) values.

Advances in molecular genetics have brought a deeper understanding of cystinuria. This autosomal recessive disease, which is caused by a defective tubular reabsorption of cystine and the three dibasic amino acids arginine, lysine and ornithine, results in a lifelong risk of renal stone formation because of the low solubility of cystine in urine. Mutations detected within the two genes known to be associated with cystinuria, SLC3A1 (related to type I) and SLC7A9 (related to non-type I), cannot, however, in all cases explain the disease. Inasmuch as a high urinary concentration of cystine is the basis of stone formation in these patients, our aim was to measure urinary total cystine, arginine, lysine and ornithine, in patients currently lacking a full genetic explanation for their disease. Thirty-three patients with cystinuria who were on long-term treatment with tiopronin or D-penicillamine were divided into two groups. Group 1 comprised eight patients who carried mutation in one of the SLC3A1 alleles and two patients who completely lacked mutations both in the SLC3A1 and the SLC7A9 genes, that is genetic findings discordant with the increased urinary excretion of cystine and the dibasic amino acids in these patients. Group 2 comprised 23 patients homozygous for mutations within SLC3A1, that is genetic findings in accordance with the excretion pattern of classic type I cystinuria. When the two groups were compared, Group 1 had a significantly higher total urinary excretion of cystine ( p<0.01) as well as of arginine, lysine and ornithine ( p<0.05) than Group 2. Also, when the two patients without mutations were excluded from the calculations, there still was a significant difference in the urinary excretion of total cystine ( p<0.05). This suggests that the two patients without any detected mutations in the two known cystine transport genes also contributed to the difference. These unexpected findings indicate that an additional gene or genes participate in the urinary cystine reabsorption in the cystinuric patients who currently are without a full genetic explanation for their disease.

Background: There is need for a reference method for transferrin glycoforms in serum to which routine immunologic methods for the alcohol marker carbohydrate-deficient transferrin (CDT) can be traceable. We describe an improved HPLC method for transferrin glycoforms., Methods: Transferrin was iron-saturated by mixing the serum with ferric nitrilotriacetic acid, and lipoproteins were precipitated with dextran sulfate and calcium chloride. Separation of glycoforms was performed on a SOURCE 15Q anion-exchange column using salt gradient elution. Quantification relied on selective absorbance of the iron-transferrin complex at 470 nm. The relative amount of each glycoform was calculated as a percentage of the area under the curve, using baseline integration., Results: The HPLC system provided reproducible separation and quantification of the asialo-, monosialo-, disialo-, trisialo-, tetrasialo-, pentasialo-, and hexasialotransferrin glycoforms. Most importantly, disialo- and trisialotransferrin were almost baseline separated. The intra- and interassay CV for disialotransferrin were <5%. Serum and the pretreated samples were stable for at least 2 days at 22 or 4 degrees C. Sera from 132 healthy controls contained [mean (SD)] 1.16 (0.25)% disialotransferrin, 4.77 (1.36)% trisialotransferrin, 80.18 (2.01)% tetrasialotransferrin, and 13.88 (1.69)% pentasialo- + hexasialotransferrin. In some cases of a high (>6%) trisialotransferrin, monosialotransferrin was detected at <0.25%. Asialotransferrin was not detected in control sera, but was detected in 57% of chronic heavy drinkers and in 62% of sera with >/=2% disialotransferrin., Conclusions: The HPLC method fulfills the requirements of a preliminary reference method for CDT and should work for any combination of serum transferrin glycoforms. This method could also be useful for confirming positive CDT results by immunoassays in medico-legal cases.

Aims/hypothesis: We aimed to evaluate how an aetiology-based classification, as recommended in the ADA and WHO guidelines for classification of diabetes mellitus, matches clinical judgement in the Diabetes Incidence Study in Sweden (DISS), a study covering incident cases of diabetic patients aged 15 to 34 years., Methods: During a 1-year period (1998), blood samples were taken at diagnosis and 4 months (median) thereafter. Patients were classified according to clinical judgement by the reporting physicians and assessments of islet antibodies (ICA, GADA, and IA-2A) and plasma C-peptide., Results: In 1998, 422 patients were registered in DISS. Among the 313 patients participating in the follow-up, most with clinical Type 1 diabetes (185/218, 85%, 95% CI 79-89%) were islet antibody positive (ab+) at diagnosis. In addition, 14 out of 58 (24%, 14-37%) with clinical Type 2 diabetes and 21 out of 37 (57%, 40-73%) with unclassifiable diabetes were antibody positive at diagnosis. Further to this, 4 out of 33 (12%, 3-28%) were antibody negative with clinical Type 1 diabetes and 4 out of 44 (9%, 3-22%) with Type 2 had converted to antibody positive at follow-up. Among those who were constantly antibody negative, 10 out of 29 (34%, 18-54%) with clinical Type 1 and 1 out of 16 (6%, 0-30%) with unclassifiable diabetes had fasting plasma C-peptide concentrations below the normal range (<0.25 nmol/l) at follow-up., Conclusion/interpretation: Most young adults with clinical Type 1 diabetes (199/218, 91%) had objective Type 1 (ab+ at diagnosis/follow-up and/or low fasting plasma C-peptide concentrations at follow-up), as did one third (18/58, 31%) with clinical Type 2 diabetes and more than half (22/37, 59%) with unclassifiable diabetes. About 10% of those who were antibody negative converted to antibody positive. Our study underlines that a classification considering aetiology is superior to clinical judgement.

Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate-protein interactions by introducing a so-called shielding reagent. Two samples from Bio-Rad (Lyphochek)--one from normal persons' blood with relatively low HbA(1c) level (HbL) and the other from diabetic patients' blood with an elevated HbA(1c) level (HbH)--were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear gradient elution with Tris. Several bands were observed on isoelectric focusing gel, which showed the same migration positions as Hb adducts, such as HbA(0), which is major Hb component containing two alpha chains and two beta chains; HbA(1c), which is post-translational glycation on the N-terminus of the beta chains of HbA(0); Foetal Hb (HbF), consisting of two alpha chains and two gamma chains; and glutathione Hb (also called HbSSG), which is the result from thiol-disulphide interchain exchange during oxidation of the thiol groups of Hb. In both HbL and HbH samples, E2 exhibited slightly higher amounts of HbF than E1. Electrospray-ionisation mass spectrometry showed that: (1) HbL-E1 was glycated with single glucose on both alpha and beta chains while no observable glycated chains were present in HbL-E2; (2) both HbH-E1 and HbH-E2 were glycated with single glucoses on both alpha and beta chains, however, compared with HbH-E1, HbH-E2 showed a higher relative intensity of the glycated beta chain and lower relative intensity of the glycated alpha chain; and (3) the degree of glycation increased with increasing glycation level of the sample. The amount of HbA(1c) presented in the eluted peaks was further determined using enzymatic digestion of glycated Hb by endoproteinase Glu-C and the subsequent separation and analysis of the digested peptides by reversed-phase high-performance liquid chromatography and capillary electrophoresis. The values of HbA(1c)/HbA(0) of the eluted peaks, i.e. HbL-E1, HbL-E2, HbH-E1 and HbH-E2, were 0.27, 0.19, 0.50 and 0.43, respectively. In both HbL and HbH samples, E1 contained higher amounts of HbA(1c) than E2. This study demonstrates the structural heterogeneity of GHb as well as the possibility of using SBAC to detect glycated species of Hb., (Copyright 2002 Elsevier Science B.V.)

Background: Third trimester fetal death is a common problem in Mozambique, occurring in approximately 5% of parturient women., Objective: To elucidate the magnitude of the gestational diabetes mellitus problem, and to estimate its prevalence in a group of women with unexplained late fetal deaths and in women with live fetuses (referents)., Methods: An incident case-referent study of 109 pregnant Mozambican women with fetal deaths and 110 women delivering liveborns, regarding fasting B-glucose, oral glucose tolerance test and glycosylated hemoglobin., Result: The difference in gestational diabetes mellitus prevalence in the two groups is not significant. The prevalence of gestational diabetes mellitus was high in both groups: 11% and 7%, respectively.

HbA1C is the stable glucose adduct to the N-terminal group of the beta-chain of HbA0. The measurement of HbA1c in human blood is most important for the long-term control of the glycaemic state in diabetic patients. Because there was no internationally agreed reference method the IFCC Working Group on HbA1c Standardization developed a reference method which is here described. In a first step haemoglobin is cleaved into peptides by the enzyme endoproteinase Glu-C, and in a second step the glycated and non-glycated N-terminal hexapeptides of the beta-chain obtained are separated and quantified by HPLC and electrospray ionisation mass spectrometry or in a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection. Both principles give identical results. HbA1c is measured as ratio between the glycated and non-glycated hexapeptides. Calibrators consisting of mixtures of highly purified HbA1c and HbA0 are used. The analytical performance of the reference method has been evaluated by an international network of reference laboratories comprising laboratories from Europe, Japan and the USA. The intercomparison studies of the network showed excellent results with intra-laboratory CVs of 0.5 to 2% and inter-laboratory CVs of 1.4 to 2.3%. Possible interferences have been carefully investigated. Due to the higher specificity of the reference method the results are lower than those generated with most of the present commercial methods which currently are calibrated with unspecific designated comparison methods. The new reference method has been approved by the member societies of the International Federation of Clinical Chemistry and Laboratory Medicine and will be the basis for the future uniform standardization of HbA1c routine assays worldwide.

Based on previous observations of the diurnal variation of urinary cystine excretion, the use of separate day and night urine collections was proposed. To improve the medical treatment of patients with cystinuria, this strategy was performed to guide the fluid intake and the administration of SH compounds (tiopronin, D-penicillamine, and MESNA).Twenty-six patients (19 treated with SH compounds and seven with alkalinization and hydration only) were followed during two 3.5-year periods. During Period 1, 24-h urine was collected and during Period 2, separate day and night urine was collected. There were 56 episodes of high urinary cystine supersaturation (> 1,200 micromol/l) during Period 2, 47% of which would have evaded detection with 24-h urine analysis. In comparison with Period 1, the urinary cystine concentration was lower (P < 0.05), and the urinary volume was higher (P < 0.05) during Period 2. Patients treated with tiopronin had reduced cystine excretion (P < 0.05) and at the end of Period 2, an increased dose of tiopronin, reflecting a more aggressive treatment. Furthermore, a reduced number of stone episodes and need of active stone removal (P < 0.05) was noted in the whole group of patients. Analyses of separate day and night urine samples can be used advantageously to reveal episodes of high supersaturation with cystine not detected in 24-h urine samples. Such a procedure might be useful for optimizing the treatment of patients with cystinuria.

For many years, urine alkalinization has been one of the cornerstones in the treatment of homozygous cystinuria. Because of the relationship found between the excretion of urinary sodium and cystine, potassium citrate has emerged as the preferred sodium-free alkalizing agent. To evaluate the usefulness of potassium citrate for urine alkalization in cystinuric patients, sodium bicarbonate and potassium citrate were compared in 14 patients (10 on tiopronin treatment and four without treatment with sulfhydryl compounds). The study started with 1 week without the use of any alkalizing agents (Period 0) followed by 2 weeks with sodium bicarbonate (Period 1) and 2 weeks with potassium citrate (Period 2). Urinary pH, volume, excretion of sodium, potassium, citrate and free cystine, as well as the plasma potassium concentration, were recorded. Potassium citrate was shown to be effective as an alkalizing agent and, in this respect, not significantly different from sodium bicarbonate. Even though a normal diet was used, a significant increase in urinary sodium excretion was observed with sodium bicarbonate (Period 1). Urinary potassium and citrate excretion increased with potassium citrate (Period 2). A significant correlation was found between urinary sodium and cystine in the tio-pronin-treated patients. No significant differences in cystine excretion were recorded in Periods 0, 1 and 2. Plasma potassium was significantly higher during Period 2, but only one patient developed a mild hyperkalemia (5.0 mmol/l). The use of potassium citrate for urine alkalization in homozygous cystinuria is effective and can be recommended in the absence of severe renal impairment.

According to the medical regulations for obtaining a driver's license in Sweden, alcohol abuse/dependency constitutes sufficient grounds for denial. In the case of a conviction for gross drunk driving, it is incumbent upon the offender to present a medical certificate verifying a "sober lifestyle". Biological markers are important tools for proving alcohol abuse in each of these contexts. In this connection, CDT analyses play a key role through their high marked specificity for increased alcohol consumption. The authors have agreed upon the guidelines as presented in this paper for determining sobriety as it pertains to possession of a driver's license. Special emphasis is placed on how CDT tests should be used and interpreted in such contexts, as well as their value as evidence in the case of increased CDT levels.

Background: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system., Methods: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the gamma- and ss-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested., Results: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma., Conclusions: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.

Background: Isoforms of transferrin interfere with measurement of carbohydrate-deficient transferrin (CDT) as a marker of heavy alcohol consumption. We evaluated the rate of inaccurate CDT results by immunoassays., Methods: We studied 2360 consecutive sera (1614 individuals) submitted for CDT assay without clinical information as well as samples from 1 patient with a congenital disorder of glycosylation (CDG Ia) and from 6 healthy carriers of CDG Ia. The CDTect, %CDT-TIA, and new %CDT immunoassays were compared with HPLC (%CDT-HPLC). Transferrin isoform pattern were evaluated by isoelectric focusing (IEF)., Results: Transferrin BC and CD heterozygotes were found at frequencies of approximately 0.7% and approximately 0.2%, respectively. Another transferrin C subtype, where di- and trisialotransferrin partly coeluted (tentatively identified as C2C3), was observed in approximately 0.6%. Compared with the %CDT-HPLC method, the immunoassays often produced low results for transferrin BC and high results for transferrin CD and "C2C3". A very high trisialotransferrin value (frequency approximately 1%) often produced high CDT immunoassay results. In four of six healthy carriers of CDG Ia, a- and disialotransferrin were highly increased and the HPLC and IEF isoform patterns were indistinguishable from those in alcohol abuse., Conclusions: Rare transferrin isoform types and abnormal amounts of trisialotransferrin (total frequency approximately 2-3%) may cause incorrect determination of CDT with immunoassays. The observed variants were readily identified by HPLC and IEF, which can be recommended for verification of CDT immunoassay results in doubtful cases. In healthy carriers of CDG Ia, CDT is high by all assays.

Capillary electrophoresis represents a relatively new analytical technique. This methodology has diversified and given rise to various modes such as capillary zone electrophoresis, capillary gel electrophoresis, micellar electrokinetic capillary chromatography, capillary isoelectric focusing and capillary isotachophoresis. If capillary electrophoresis was first introduced in research laboratories, this technique is now making an entrance to the clinical laboratory. This is due to its rapid and high-efficiency separation power, its potential applications and its possible automation. Thus, capillary electrophoresis represents an attractive alternative to some time-consuming techniques. Thanks to its versatility, the use of capillary electrophoresis has been proposed for the separation and quantification of a wide spectrum of biological components ranging from macromolecules (proteins, lipoproteins, nucleic acids) to small analytes (amino acids, organic acids or drugs). This paper illustrates the potential of capillary electrophoresis which should rapidly become a major technology for a modern clinical laboratory.

Objectives: To examine the relationship between previous glycaemic exposure and prevalence of retinopathy 8 years after diagnosis of diabetes in 58 islet cell antibodies (ICA)-negative noninsulin-dependent diabetes mellitus (NIDDM) patients and in a group of 14 ICA-positive 'NIDDM' and insulin-dependent diabetes mellitus (IDDM) patients., Design and Methods: The Wisconsin retinopathy scale was used to assess the retinopathy which was graded into mild, moderate and severe nonproliferative diabetic retinopathy (NPDR), or proliferative retinopathy (PDR). The frequency and severity of retinopathy was related to HbA1c levels at diagnosis, and 3 and 5 years later., Results: Thirty of the 58 ICA-negative NIDDM patients (52%) but only 2 of the 14 ICA-positive 'NIDDM' or IDDM patients (14%) had mild-moderate-severe NPDR 8 years after diagnosis (P = 0.02). None had PDR. Retinopathy 8 years after diagnosis in NIDDM (= 58 ICA-negative patients) was correlated with the degree of glycaemic control (HbA1c levels) at 3 and 5 years after diagnosis, but not to HbA1c levels at diagnosis. The relative risk for a higher average HbA1c (per percentage) at 3 and 5 years was 1.56 for any retinopathy vs. no retinopathy (95% confidence interval 1.1-2.2; P = 0.01) and 1.68 for moderate to severe NPDR in comparison with no DR and mild NPDR (95% confidence interval 1.0-2.8; P = 0.04)., Conclusions: Retinopathy after 8 years of diabetes in NIDDM patients was associated with impaired glycaemic control during previous years but not with glycaemic control at baseline. Good glycaemic control may prevent retinopathy in patients with NIDDM.

In Tanzania, assessment of blood glucose is the most frequently used method for evaluating glycaemic control in diabetic patients. The patients' metabolic control is often poor and the determination of glycated haemoglobin (HbA1c) for long-term control could be a valuable tool to better manage the treatment. The aim of this study was to determine whether an immediate assessment method for HbA1c (DCA 2000 analyzer) gives reliable results in the warm and moist climate of east Africa. The study was performed in two parts. One equipment test in Sweden where blood samples from 65 diabetic patients were analysed in a DCA 2000 kept at room temperature and in another kept in a climate chamber. The samples were also analysed with HPLC as a reference method. In the second part HbA1c was analysed in 159 Tanzanian diabetic patients with a DCA 2000 and with HPLC combined with a filter paper technique (HbA1c via Post). The study showed that the DCA 2000 gives reliable results at 85% humidity and a temperature not exceeding +31 degrees C. The correlation with the HPLC analysis varied between 0.94 and 0.98. The conclusion is that the DCA 2000 analyzer can be used in Tanzania during the winter but has to be placed in an air-conditioned room if the temperature exceeds +31 degrees C.

We prepared a candidate primary reference material for the forthcoming international standardisation of beta-N-terminal glycated hemoglobin A measurements. It consists of well-defined mixtures of purified beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A. First, beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A were isolated, purified to homogeneity, and characterised. The techniques used were cation exchange and affinity chromatography for the purification, and high performance liquid chromatography, capillary isoelectric focusing, electrospray ionisation mass spectrometry, and peptide mapping for the characterisation. Hemoglobins from blood of healthy, non-diabetic volunteers were obtained with a purity of > 99.5% for non-glycated hemoglobin A and of > 98.5% for beta-N-terminal glycated hemoglobin A. However, results from peptide mapping indicate that the beta-N-terminal glycated hemoglobin A preparations still contain some non-beta-N-terminal glycated hemoglobins, co-eluting with beta-N-terminal glycated hemoglobin A. The exact content of beta-N-terminal glycated hemoglobin A in these preparations could be determined by a procedure consisting of standard addition, enzymatic cleavage and quantification of the resulting beta-N-terminal peptides to be in the range from 95-97.5%. Since the beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A content could be exactly determined in the materials prepared, mixtures of both components could be successfully used to calibrate the candidate reference methods.

The article reports findings in a study of 198 subjects attending a psychiatric out-patient clinic, with known or suspected high alcohol consumption during a 12-month period, about half of whom had had their driving licences revoked. The level of carbohydrate-deficient transferrin (CDT) was found to be a valuable marker of alcohol consumption, and a useful adjunct to the measurement of liver enzymes. Both GGT (gamma-glutamyltransferase) and CDT levels were significantly higher in high alcohol consumers than in low consumers. Alcohol the two markers did not differ from each other in statistical significance, CDT was associated with greater sensitivity and specificity; the sensitivity of CDT was 69% for men and 79% for women, as compared with 62% and 40%, respectively, for GGT; the specificity of CDT was 81% for men and 100% for women, as compared with 82% and 72%, respectively, for GGT. Together, GGT and CDT detected 91% of the male and 93% of the female high consumers. Among younger men, CDT values were higher in the subgroup with a history of traffic offences than in the subgroup without such a history, thus suggesting that CDT levels may be increased by heavy weekend beer consumption. A few cases of false-positive CDT results were found to be attributable to genetic anomalies of the transferrin molecule. Cases characterised by disparity between the CDT level and the clinical picture require further, more specific, analysis. Used in combination with GGT, CDT is thus a feasible marker for use in monitoring alcohol consumption in drivers needing to qualify for the restoration of their licences.

We describe here an in vitro effect of lithocholic acid (LA), a secondary, hydrophobic bile acid, on the rate of polymerization of mutant, Z and wild-type, M alpha-1-antitrypsin (AAT). Using thioflavine T fluorescence and turbidity assays we demonstrated that the rate of aggregation for the Z AAT in the presence of LA at a molar ratio of 1:5 AAT to LA, in Tris-buffered saline, pH 7.4, is at least twice that of the Z protein alone or the M variant with and without LA. Also, Z AAT incubated for 48 h at room temperature had more than 50% diminished antielastase activity, while M AAT had only a 25% reduction in activity. Analysis of the AAT and AAT-LA samples after cleavage with pancreatic elastase by SDS-PAGE 10% gels showed that interaction between Z or M AAT and LA abolishes their ability to form SDS stable complexes with an enzyme and both of these forms of AAT showed elastase substrate behavior. Furthermore, Z as well as M AAT incubated with LA at 41 degrees C and cleaved with elastase showed only 80 to 60% increased thermal stability compared to 100% stabilization for the cleaved AAT alone in the absence of LA. These observations suggest that a rearrangement of the AAT molecule as a result of interactions with LA increases aggregation of AAT and diminishes its inhibitory activity., (Copyright 1998 Academic Press.)

Patients with fragility fractures have low bone mineral density (BMD)--this statement is supported mainly by data on women. In this study, including only men, the objectives were to determine whether a decline in BMD alone or in combination with data on male sex hormones and skinfold thickness could be of value in predicting forthcoming fractures. We also wanted to find out whether high consumers of alcohol can be identified by measuring BMDs and male sex hormones. A prospective, population-based study was performed in the city of Malmö, Sweden. 242 men were randomly selected; all were of Scandinavian ethnic background, and were aged 50, 60, 70, and 80 years. Forearm BMD, testosterone, sex-hormone-binding globulin (SHBG), and skinfold thickness were analyzed. In addition, alcohol consumption and carbohydrate-deficient transferrin (CDT)--a marker of alcohol abuse--were analyzed. The study group was followed prospectively for 7 years and all fractures sustained were recorded. Prospectively, for a 1 SD decrease in forearm BMD, the Cox proportional hazard model gave a relative risk (RR) of 1.75 with a 95% confidence interval of 1.08-2.83 for a forthcoming fracture and 3.88 (1.30-11.57) for a hip fracture. For a 1 SD change in skinfold thickness, measured on the dorsum of the hand, a RR of 1.69 (0.99-2.87) for a forthcoming fracture was found and the corresponding value for hip fracture was 2.34 (1.10-5.00). Testosterone and SHBG did not enhance fracture prediction. Abusers of alcohol had, retrospectively, significantly more fractures. Individuals with alcohol consumption rates in the highest quartile had significantly higher CDT levels, but we were unable to identify high consumers of alcohol by analyzing BMD or sex hormones. In this study we found that forearm BMD and skinfold thickness could be used in predicting forthcoming fractures in men.

A reference method that specifically measures hemoglobin (Hb) A1c is an essential part of the reference system for the international standardization of Hb A1c/glycohemoglobin. We have developed a new method for quantification, based on the specific N-terminal residue of the hemoglobin beta-chains. Enzymatic cleavage of the intact hemoglobin molecule with endoproteinase Glu-C has been optimized to obtain the beta-N-terminal hexapeptides of Hb A1c and Hb A0. These peptides have been separated by reversed-phase HPLC and quantitated by electrospray ionization-mass spectrometry (method A) or by capillary electrophoresis (method B). With these peptides and hyphenated separation techniques, it has been possible to overcome the insufficient resolution of currently used protein separation systems for Hb A1c.

Objective: To facilitate HbA1c determination, we evaluated an HbA1c filter paper system enabling capillary blood sampling at home by the patients., Research Design and Methods: Capillary blood (two drops) was applied to a filter paper (HbA1c Via Post) and sent to the laboratory where a small disc was punched out on the filter paper. Hemoglobin was eluted from the disc in a buffer containing cysteine to eliminate the interfering glutathione adduct (HbA3) formed during storage. Analysis was performed by ion-exchange chromatography (Mono S, high-performance liquid chromatography), and the eluate was compared with hemolysate of venous blood from 41 patients. The stability of blood impregnated on filter paper was checked at different temperatures over different periods of time., Results: There was an excellent agreement (r = 0.99) between HbA1c values from capillary blood on filter paper and HbA1c values from venous blood. HbA1c values were constant when stored on filter paper for 5-7 days at 20-21 degrees C (room temperature) or at 4-6 degrees C (refrigerator) for 10 days as well as at -70 degrees C for several months after blood sampling. A new chromatographic-interfering hemoglobin fraction both from venous and capillary samples was identified as free alpha-chain of hemoglobin., Conclusions: The HbA1c filter paper system enables capillary blood sampling at home, eliminates the need of vein puncture in children and adults, and provides the diabetologist with an HbA1c value when the patient visits the clinic without a need for a previsit phlebotomy.

Normal human serum transferrin is present in several isoforms, due to differences in glycosylation. Transferrin has two potential glycosylation sites, both normally occupied by oligosaccharide chains. Two of the transferrin isoforms, called carbohydrate deficient transferrin, are specifically increased in patients with high alcohol consumption. In this study, five isoforms of transferrin were isolated from patients with high alcohol consumption. N-linked glycans were released by N-glycosidase digestion and were radioactively labeled by NaB3H4 reduction. The purified oligosaccharides were analyzed by high-pH anion-exchange chromatography, and the carbohydrate composition of each individual transferrin isoform was determined. The carbohydrate deficient transferrin isoforms were found to lack one or both of their entire carbohydrate chains.

The formation of stones in patients with cystinuria can be counteracted by reducing the urinary concentration of cystine and by increasing its solubility. Thirty-one patients with homozygous cystinuria and treated with tiopronin (2-mercaptopropionylglycine) were followed for between 0.4 and 12 years (median 8.8). With the aim of avoiding cystine concentrations above 1200 mumol/l, the daily dose varied between 500 and 3000 mg (median 1500). The therapeutic effect was evaluated from the clinical symptoms and repeated radiographic examinations. The rate of stone formation during the treatment period was reduced by 60% in comparison with the pretreatment period (P < 0.001). The frequency of active stone removal was reduced by 72% (P < 0.05). The formation of new stones was associated with a higher cystine concentration than was the case during periods when stone formation and stone growth were excluded (P < 0.05). The probability of new stone formation increased with increasing concentrations of cystine up to 1100 mumol/l, but stone formation was not accentuated above 1200 mumol/l. There was no significant relationship between the 24 h excretion of cystine and stone formation. It is concluded that the formation of cystine stones can be efficiently counteracted during treatment with tiopronin, guided by analysis of the concentration of urinary cystine.

As with many other amino acids the transport of cystine across the tubular epithelium is coupled to a parallel transport of sodium. We have studied the effect of a sodium-restricted diet on the urinary excretion of cystine in 13 patients with cystinuria, 7 of whom were treated with the SH compound tiopronin (2-mercaptopropionylglycine). Five of the patients with tiopronin and 5 without were also given sodium bicarbonate. The patients were instructed to follow a sodium-restricted diet during three periods of 2 weeks each. Four levels of sodium intake were obtained including the preexperimental unrestricted diet. The average 24-hour excretion of free cystine increased by 3.1 mumol (0.75 mg) for each millimole increase in urinary sodium (p < 0.001). There was a greater sodium-related increase in excretion of cystine among patients without tiopronin treatment compared with the group with tiopronin (p < 0.01). Withdrawal of sodium bicarbonate resulted in a decrease in the 24-hour cystine excretion (p < 0.05). In the patients treated with tiopronin the excretion of the mixed disulfide increased with increasing urinary sodium (p < 0.05) suggesting a sodium-dependent active tubular reabsorption of this compound as well. We conclude that in spite of a defective proximal tubular reabsorption of cystine in cystinuria the reabsorption can be increased by restricting the intake of sodium. This effect of sodium may have clinical consequences for some cystinuric patients.

In an attempt to improve the diagnostic value of urine analysis in patients with homozygous cystinuria, we studied the diurnal variation in urine composition. A simplified estimate of the ion-activity product of cystine was used to increase the probability of identifying patients with a particular risk of stone formation. Eight 6-h urine samples were collected during two 24-h periods. The highest urinary excretion of cystine was recorded between 1200 and 1800 hours and the lowest between 0000 and 0600 hours, whereas the urinary cystine concentration was highest between 0000 and 0006 hours and lowest between 1200 and 1800 hours. The approximate ion-activity product of cystine had a maximal level between 0000 and 0600 hours but a minimal level between 0600 and 1200 hours. The differences between different periods were numerically more pronounced in terms of the ion-activity product of cystine than in terms of concentration. The peak concentrations of cystine in 6-h samples were about 90% higher than the corresponding concentrations in 24-h urine samples. It is concluded that the analysis of cystine in 6-h urine samples reveals transient episodes of cystine supersaturation that otherwise will remain undetected. Further studies are, however, needed to establish its usefulness in clinical practice.

We report the results of a biochemical evaluation of long-term treatment of cystinuria with the SH compound tiopronin (2-mercaptopropionylglycine). The effects of tiopronin were studied by monitoring the urinary excretion of free cysteine and the mixed disulfide between tiopronin and cysteine. Thirty-one patients with homozygous cystinuria were treated with tiopronin for 0.4-12 years (mean 7.8 years). The urinary concentration of free cysteine was used to adjust the tiopronin dose. In 28 of the 31 patients a mean urinary cystine concentration of less than 1,200 mumol/1(288 mg/l) was achieved with the final dose. The final daily doses of tiopronin ranged from 250 mg (1.5 mmol) to 3,000 mg (18.4 mmol; mean 1,540 mg; 9.4 mmol). In a majority of the patients the treatment reduced the 24-hour urinary free cystine excretion effectively, on average by 0.61 mumol (0.15 mg)/mg of tiopronin administered. No changes in the efficacy of tiopronin over time were observed, and the frequency of adverse effects was acceptable. To evaluate the effects of tiopronin on the metabolism of cystine we calculated the total urinary excretion of cystine as the sum of free cystine and the amount of cystine corresponding to the cysteine content of the tiopronin-cysteine disulfide. At low doses of tiopronin there was an increase in urinary excretion of the mixed disulfide as well as of total cystine. Monitoring urinary cystine concentration is necessary to achieve adequate individualized doses of tiopronin. Assessment of the mixed tiopronin-cysteine disulfide and the urinary excretion of total cystine shows that tiopronin may interfere with cystine metabolism in a more complex way than through a simple disulfide exchange reaction with urinary cystine.

The Pharmacia Mono S column for glycated haemoglobin (HbA1c) was compared with Mono S HR 5/5 and three other cation exchange columns to optimize analysis of HbA1c. This column allows a significant decrease in separation time, resulting in a sample throughput of 12 samples per hour. The choice of parameters for integration of peak areas in the chromatograms is very important for reproducible results. Since pure calibrators of HbA1c are not available, the relative contribution of HbA1c to the total amount of haemoglobin is calculated. We have compared valley-to-valley integration with baseline integration to assess reproducibility. Valley-to-valley integration significantly enhances the reproducibility of the method and is therefore preferable for routine analyses of HbA1c.

We developed a new fully automated ion-exchange chromatographic method for quantitating carbohydrate-deficient transferrin (CDT) on a Mono Q column. Quantitation relies on the selective absorbance of the iron-transferrin complex at 460 nm. Transferrin isoforms deficient in sialic acid, with pIs 5.7 and 5.9, can easily be separated and quantitated as a percentage of the total transferrin. This method has been applied to samples from teetotalers, occasional drinkers, patients with recent heavy alcohol consumption, and patients during detoxification. The sensitivity of the method was 55% in patients reporting 40-70 g daily ethanol consumption and nearly 100% in heavily intoxicated patients (70-500 g daily consumption). The half-life of the dominating pI 5.7 isoform in this group was 9.5 (+/- 1) days during detoxification. A CDT value > 0.8% is a highly specific marker for alcohol abuse and is greatly superior to other currently available biological markers.

We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive time-resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-amplified nucleic acid fragment. The hybrids formed were then collected onto streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy underlying the design of the Eu-labeled ASO probes for the solution hybridization assay. The method was applied to the detection of the Z-mutation in the alpha 1-antitrypsin gene. Evaluation of whole-blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.

Here we report the occurrence of five different beta chain hemoglobin variants not previously described in Sweden. The variants were found during quantification of HbA1c using ion exchange high performance liquid chromatography (HPLC) or isoelectrofocusing. Samples were examined either at protein level by separation of globin chains on C8 reversed phase HPLC, digestion with trypsin or lysylendopeptidase and separation of peptides by C18 reversed phase HPLC, or at DNA level by direct nucleotide sequencing of double-stranded DNA fragments amplified from exon 1 + 2 of the beta-globin gene. The variants were: Hb Raleigh [beta 1 (NA1)Val-->Ac-Ala], Hb J-Baltimore [beta 16(A13)Gly-->Asp], Hb Tacoma [beta 30(B12)Arg-->Ser], Hb K-Ibadan [beta 46(CD5)Gly-->Glu], and Hb Fukuyama [beta 77(EF1)His-->Tyr]. Hb Tacoma, Hb K-Ibadan, and Hb Fukuyama were slightly unstable in the isopropanol test, but no signs of hemolysis were found in the patients who all had normal hematological findings.

The 24-h urine excretion of 20 amino acids was investigated in 24 cystinuric and 15 normal dogs. The diagnosis of cystinuria was based on infrared spectroscopy of removed uroliths, which in all cases were composed of pure cystine. Seven of 24 cystinuric dogs showed normal cystine excretion compared to normal dogs, and four of 24 dogs showed normal total amino acid excretion. In contrast to earlier investigations, almost half of the cystinuric dogs (46%) showed elevated excretion of five or more amino acids. Isolated cystinuria, or isolated dibasic amino aciduria was not found. Compared to normal dogs, the cystinuric dogs showed a significantly (P < 0.05) increased excretion of cystine, arginine, lysine, cystathionine, glutamic acid, threonine and glutamine. There was a significant correlation (P < 0.05) between the urinary excretion of cystine and 10 other amino acids, with the highest correlation found (P < 0.001) for arginine, lysine, cystathionine, ornithine and 1-methyl-histidine. Three patterns of amino acid excretion could be identified: (1) increased excretion and a significant correlation with cystine for the three dibasic amino acids (lysine, arginine and ornithine), compatible with a common reabsorption mechanism as shown in man. This pattern was also found for cystathionine and glutamic acid, which might indicate a relation in metabolism or transport; (2) increased excretion but no correlation with cystine for glutamine, threonine and citrulline; (3) good correlation with cystine, but no increased excretion for 1-methyl-histidine, phenylalanine, 3-methyl-histidine, leucine and alanine. The great variation in urinary cystine excretion suggests that factors other than the excretion of cystine must be considered as causes of cystine urolith formation. For example, cystinuric dogs were found to have lower diuresis than normal dogs and produced urine with higher cystine concentration thereby increasing the risk of cystine urolith formation.

A clinical study covering 1 to 6 years was undertaken during which 25 cystinuric dogs were orally treated with 2-mercaptopropionylglycine (2-MPG). The drug was effective at dissolving uroliths at a dose of approximately 40 mg kg-1 body weight. In 15 dogs with bladder uroliths, complete urolith dissolution was achieved on 9/17 occasions (53%). When 2-MPG was administered prophylactically at 30 mg/kg body weight, uroliths did not reform in 14 dogs (56%). In four dogs, uroliths re-formed during treatment, but dissolved when the dose of 2-MPG was raised to 40 mg kg-1 body weight. Six dogs were surgically treated, and in two of these animals the uroliths were found to consist of magnesium ammonium phosphate. Euthanasia was performed on six dogs during the study; three because of recurrent uroliths with urethral obstruction, and three because of aging. In one dog, uroliths were present in the bladder throughout the study. The purpose of the study was to propose a new strategy for individual treatment of cystinuric dogs. This was accomplished by measuring the urinary free cystine concentration and the mixed cysteine-2-MPG disulphide in a subgroup of 15 of the 25 dogs. To evaluate cystine excretion, morning samples of urine were used, and the cystine concentration was related to the creatinine concentration. For dose adjustment it was difficult to evaluate the effect of 2-MPG on urinary cystine excretion, especially when cystine uroliths were present. However, this variable was studied in order to identify dogs with a strong tendency for urolith formation during 2-MPG treatment. In some cases, urinary cystine excretion returned to normal with time, and in three dogs, 2-MPG treatment could be stopped after 1.5 to 3.5 years. In spite of no further treatment, urinary cystine was almost undetectable up to 2 years later, and the dogs did not develop any new uroliths. It was concluded that 2-MPG is a satisfactory alternative treatment for cystinuric dogs. It has a good prophylactic effect, shown as a change in the rate of urolith formation from on average 6 months before to 17 months during 2-MPT treatment. The drug was shown to have few side effects, and the dog owner drug compliance can be followed by measurement of the mixed 2-MPG-cysteine disulphide.

In this method for automatically running and staining isoelectric focusing (IEF) gels, pre-made dehydrated polyacrylamide gels were rehydrated before assays run with the PhastSystem (Pharmacia LKB Biotechnology). The typing of genetic variants of hemoglobin and alpha 1-antitrypsin in narrow pH gradients (pH 6.7-7.7 and 4.2-4.9, respectively) was simple, convenient, and reproducible. The clinically important variants of alpha 1-antitrypsin (ZZ and SZ) were identified from serum or dried blood on filter paper. The fast screening of abnormal hemoglobin samples (HbS) for cases in clinical medicine was easily performed. The total analysis time for the phenotyping with conventional protein staining was approximately 60 min.

From liquefied human seminal plasma, we purified the predominant basic protein which appears following liquefaction of coagulated semen. The protein was purified in the presence of di-isopropylfluorophosphate to retard its degradation. Heparin-Sepharose chromatography was followed by gel filtration (Biogel P 60) and by fast performance liquid chromatography on a reversed phase column (C8). The basic protein is a single polypeptide chain with an apparent molecular mass of 12.8 kDa, and has a pI value between that of trypsinogen (9.3) and cytochrome C (10.3). The protein contains no carbohydrate, is rich in histidine, glutamate, and lysine, but is devoid of both cysteine and methionine. The amino-terminal portion of the protein sequence is unique: H N K Q E G R D H D K S K G H F H R V V I H H K G G K A H R G-. A specific rabbit antiserum was raised against the 12.8 kDa basic protein. The protein was found to be unique to seminal plasma among all extracellular fluids examined. Three immunologically related 52 kDa, 71 kDa, and 76 kDa proteins were identified in seminal vesicle secretion when it had been reduced. Prostatic enzyme(s) degraded these proteins to the 12.8 kDa basic protein and several other basic proteins with apparent molecular masses below 18 kDa.

Glucagon was iodinated with the lactoperoxidase method at pH 10.0 in the presence of propylene glycol using a substitution of 0.3 g-atom I/mol glucagon. Under these conditions the reactivity of the iodine to tyrosine at position 13 is found to be 4-fold that of the tyrosine at position 10. The amount of diiodotyrosine was less than one-twentieth that of the monoiodotyrosine at either tyrosine residue. Relatively pure monoiodo[125I]tyrosine-13-glucagon can be separated from other iodoglucagons by means of DEAE-chromatography. Such a homogeneous preparation with a known position of the iodine makes it possible to study a specific interaction between the monoiodoglucagon and the glucagon antisera or the glucagon receptor.

A young woman with a previous history of anorexia nervosa presented with severe finger clubbing. Urine samples intermittently contained significant amounts of aspartylglucosamine. Liver biopsy showed abnormal cytoplasmic inclusions in phagocytic cells. The patient was found to be abusing senna laxative.