| 1. |
- Lundberg, Peter, et al.
(författare)
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1H NMR determination of urinary betaine in patients with premature vascular disease and mild homocysteinemia
- 1995
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 41:2, s. 275-283
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Tidskriftsartikel (refereegranskat)abstract
- Urinary N,N,N-trimethylglycine (betaine) and N,N-dimethylglycine (DMG) have been identified and quantified for clinical purposes by proton nuclear magnetic resonance (1H NMR) measurement in previous studies. We have assessed these procedures by using both one-dimensional (1-D) and 2-D NMR spectroscopy, together with pH titration of urinary extracts to help assign 1H NMR spectral peaks. The betaine calibration curve linearity was excellent (r = 0.997, P = 0.0001) over the concentration range 0.2-1.2 mmol/L, and CVs for replicate betaine analyses ranged from 7% (n = 10) at the lowest concentration to 1% (n = 9) at the highest. The detection limit for betaine was < 15 mumol/L. Urinary DMG concentrations were substantially lower than those of betaine. Urinary betaine and DMG concentrations measured by 1H NMR spectroscopy from 13 patients with premature vascular disease and 17 normal controls provided clinically pertinent data. We conclude that 1H NMR provides unique advantages as a research tool for determination of urinary betaine and DMG concentrations.
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| 2. |
- Berg, Cecilia, 1969-, et al.
(författare)
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Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides.
- 1995
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 41:10, s. 1461-6
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Tidskriftsartikel (refereegranskat)abstract
- Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.
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| 3. |
- Dizdar (Dizdar Segrell), Nil, et al.
(författare)
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Human pharmacokinetics of L-3,4-dihydroxyphenylalanine studied with microdialysis
- 1999
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 45:10, s. 1813-1820
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Tidskriftsartikel (refereegranskat)abstract
- Background: Intravenous and subcutaneous microdialysis was performedto compare the free concentrations and pharmacokinetics of L-3,4-dihyroxyphenylalanine(L-dopa) in blood and tissue in healthy subjects and in patientswith Parkinson disease.Methods: Nine healthy volunteers and 10 patients with Parkinson disease, stage 1.5–2 according to the Hoehn-Yahr rating scale, took part of the study. In the patient group subcutaneous microdialysis and ordinary blood sampling were performed, whereas in the control group intravenous microdialysis was also performed. Microdialysis samples were collected in fractions of 15 min. The first two fractions were collected for analysis of basal concentrations. A blood sample was also taken. The patients were then given one tablet of Madopar® (100 mg of L-dopa and 25 mg of benserazide),and the microdialysis was continued for another 210 min. Bloodsamples were obtained at 30-min intervals.Results: The serum samples gave a significantly higher meanarea under the curve (AUC; 491 ± 139 µmol ·min/L) than that for intravenous dialysates (235 ± 55.3µmol · min/L), suggesting a protein binding of50%. The L-dopa concentrations from the subcutaneous dialysatesmatched those from the intravenous dialysates, indicating rapiddistribution of L-dopa to the tissues.Conclusions: Parkinsonian patients in early stages of the disease have a pharmacokinetic pattern of free L-dopa similar to that of healthy subjects. Comparison of AUCs from microdialysis with ordinary serum analysis revealed data indicating significant protein binding. Microdialysis is a suitable and easily applied tool in pharmacokinetic studies.
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| 4. |
- Hietala, M, et al.
(författare)
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DNA-based carrier screening in primary healthcare : screening for aspartylglucosaminuria mutations in maternity health offices
- 1996
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 42:9, s. 1398-1404
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Tidskriftsartikel (refereegranskat)abstract
- Large-scale genetic screening programs are complex enterprises in which ethical, technical, medical, and socioeconomic aspects have to be handled with professional expertise. Establishment of automated, relatively robust, and inexpensive laboratory techniques is one step of this path. Here a pilot carrier-screening program for the mutations causing aspartylglucosaminuria was carried out for pregnant women in primary care maternity health offices. Women (1975) were tested before their 12th week of pregnancy, and 31 heterozygotes were detected. The sampling was based on dried blood strips, facilitating convenient handling and inexpensive mailing to the laboratory. The mutation detection technique, solid-phase mini-sequencing simplified by the use of scintillation microplates and automated equipment, proved to be rapid, simple, inexpensive, and reliable, with a low repeat rate (2.5%). In conclusion, we found that good collaboration between the primary healthcare unit, the laboratory, and counseling experts, combined with modern laboratory technology, facilitate reliable low-cost genetic testing.
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| 5. |
- Kronstrand, Robert, et al.
(författare)
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Codeine Concentration in Hair after Oral Administration Is Dependent on Melanin Content
- 1999
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 45:9, s. 1485-1494
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Tidskriftsartikel (refereegranskat)abstract
- Background: Analysis of drugs in hair has been used on a qualitative basis to estimate earlier exposure to drugs. Clinical applications are rare because of the lack of dose–response relationships in the studies performed to date, and questions remain regarding the mechanisms of drug incorporation into hair. Several human studies have shown differences in drug accumulation between pigmented and nonpigmented hair. However, the melanin concentration in hair was not determined and correlated to the amount of drug incorporated.Methods: Nine human subjects were given codeine as a single oral dose, and plasma codeine concentrations were determined for 24 h, using gas chromatography–mass spectrometry. Hair samples were obtained weekly for a month. Total melanin, eumelanin, and codeine were measured quantitatively in hair samples by spectrophotometry, HPLC, and gas chromatography–mass spectrometry, respectively.Results: There was an exponential relationship between codeine and melanin concentrations in hair, (r2 = 0.95 with total melanin and r2 = 0.83 with eumelanin). After normalizing the results by the area under the curve for codeine in plasma, we obtained r2 = 0.86 for codeine vs total melanin and r2 = 0.90 vs eumelanin.Conclusions: Our results stress the importance of melanin determination when measuring drugs in hair. We postulate that analysis of drug concentration in hair may be worthwhile in the monitoring of drug compliance if the results are normalized for melanin content.
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| 6. |
- Pastinen, T, et al.
(författare)
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Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation
- 1996
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 42:9, s. 1391-1397
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Tidskriftsartikel (refereegranskat)abstract
- We developed a multiplex, solid-phase minisequencing method to detect multiple single-nucleotide polymorphisms in an undivided sample. The amplified DNA templates are first captured on a manifold. Then, with multiple minisequencing primers of various sizes, single-nucleotide extension reactions are carried out simultaneously with fluorescently labeled dideoxynucleotides. The size of the extended product, determined by using a DNA sequencing instrument, defines the site of the polymorphisms, and the incorporated nucleotide gives the identity of the nucleotide at each site. HLA-DQA1 typing was used as a model system to evaluate the method. The DR2 subgroup of the HLA-DRB1 gene was typed along with the DQA1 gene to demonstrate the feasibility of the method in analyzing multiple genes at multiple sites simultaneously. The method is generally applicable for screening any single-nucleotide polymorphisms or point mutations, and its manifold format allows practical handling of large numbers of samples.
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| 7. |
- Paunio, T, et al.
(författare)
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Preimplantation diagnosis by whole-genome amplification, PCR amplification, and solid-phase minisequencing of blastomere DNA
- 1996
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 42:9, s. 1382-1390
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a new method for preimplantation diagnosis of inherited diseases. Our procedure for the identification of point mutations in single cells combines whole-genome amplification using 15-mer random primers (primer extension preamplification, PEP) with a single locus-specific PCR amplification, followed by detection of the mutation by solid-phase minisequencing. The procedure was evaluated by detecting three disease-causing mutations and seven polymorphic nucleotides located on different human chromosomes from single granuloma and blastomere cells. The correct genotype of the cell was identified at 96% of the nucleotide positions analyzed, showing that a representative part of the genome is amplified during PEP. We estimate that PEP yielded at least 1000 copies of the genome. The quantitative nature of the solid-phase minisequencing method allowed us to notice that preferential amplification of one allele occurs at heterozygous loci during PEP, which is a potential problem in preimplantation diagnosis.
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| 8. |
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| 9. |
- Rånby, Mats, et al.
(författare)
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Isocitrate as Calcium Ion Activity Buffer in Coagulation Assays
- 1999
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 45:8, s. 1176-1180
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ca2+ activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca2+ activity at ∼1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca2+ buffering improves diagnostic efficacy in global diagnostic coagulation tests.Methods: Buffering was investigated by mixing CaCl2 and 11 candidate compounds and determining Ca2+ activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca2+ activity to ∼1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved.Results: Two suitable Ca2+ buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca2+ buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca2+ activity of 1.60 ± 0.07 mmol/L, compared with 2.73 ± 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 ± 5.7, compared with 3.6 ± 5.0 (P <0.005) for the original APTT.Conclusions: Isocitrate can be used to buffer Ca2+ activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca2+ activity shows signs of improved diagnostic efficacy.
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| 10. |
- Turner, APF, et al.
(författare)
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In vitro diagnostics in diabetes: Meeting the challenge
- 1999
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Ingår i: Clinical Chemistry. - : American Association for Clinical Chemistry; 1999. - 0009-9147 .- 1530-8561. ; 45:9, s. 1596-1601
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Tidskriftsartikel (refereegranskat)abstract
- Diabetes is one of the leading causes of death and disability in the world. There is a large population in the world suffering from this disease, and the healthcare costs increase every year. It is a chronic disorder resulting from insulin deficiency and hyperglycemia and has a high risk of development of complications for the eyes, kidneys, peripheral nerves, heart, and blood vessels. Quick diagnosis and early prevention are critical for the control of the disease status. Traditional biosensors such as glucose meters and glycohemoglobin test kits are widely used in vitro for this purpose because they are the two major indicators directly involved in diabetes diagnosis and long-term management. The market size and huge demand for these tests make it a model disease to develop new approaches to biosensors. In this review, we briefly summarize the principles of biosensors, the current commercial devices available for glucose and glycohemoglobin measurements, and the recent work in the area of artificial receptors and the potential for the development of new devices for diabetes specifically connected with in vitro monitoring of glucose and glycohemoglobin HbA(1c). (C) 1999 American Association for Clinical Chemistry.
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