A comparative study of normal and reverse phase high pressure liquid chromatography for analysis of porphyrins accumulated after 5-aminolaevulinic acid treatment of colon adenocarcinoma cells.

Primary adenocarcinoma cells of the rectosigmoid colon (WiDr-cells) were treated with 5-aminolevulinic acid (5-ALA). Cellular porphyrins were separated and quantified by high performance liquid chromatography (HPLC), both as free porphyrin acids after an easy extraction method with a subsequent reverse phase technique, and then as porphyrin esters after a more laborious extraction method and subsequent normal phase technique. The porphyrins were detected by means of a fluorescence detector. Analysis by normal phase HPLC indicated that 81% (739 pmol/mg protein) of the total amounts of fluorescing porphyrins accumulated was protoporphyrin IX, while similar analysis by reverse phase HPLC indicated that PpIX constituted 91% (622 pmol/mg protein) of the accumulated porphyrins. In addition to protoporhyrin IX, copro-, hexa-, hepta- and uroporphyrins were observed in extracts from 5-ALA-treated cells by both methods. The discrepancy between the two methods increased with increasing hydrophilicity of the analysed porphyrins, with uroporphyrin estimated to be 6-fold higher (63 vs. 10 pmol/mg protein) by normal than by reverse phase HPLC.

The concentration of high molecular weight compounds in interstitial tissue fluid: a study in patients with post-reconstructive leg oedema.

OBJECTIVES: To study whether relative changes in the concentration of different proteins of the subcutaneous interstitial tissue fluid in patients with postreconstructive leg oedema could give information on the integrity of the capillary wall following vascular reconstruction. MATERIALS: Sixteen patients developed ipsilateral leg oedema following femoropopliteal (n = 14) or femorotibial (n = 2) bypass surgery for intermittent claudication (n = 10) or critical lower limb ischaemia (n = 6) caused by obliterating atherosclerosis. METHODS: The leg volume increase was measured according to the formula of a truncated cone. The blister suction method was used for sampling of subcutaneous interstitial tissue fluid. The concentration of albumin, transferrin, immunoglobulin G and alpha 2-macroglobulin of serum and blister fluid was measured with nephelometry. RESULTS: Seven days postoperatively the mean leg volume increase was 32% (19.8%) in the operated leg compared to the contralateral side (p < 0.01). In the blister fluid each of the proteins had a significantly higher concentration in the operated leg compared to the control leg. This concentration difference was relatively larger for the proteins with the highest molecular weight, immunoglobulin G (p = 0.006) and alpha 2-macroglobulin (p = 0.002). CONCLUSIONS: A relatively larger concentration difference of the bigger molecules in the subcutaneous interstitial tissue fluid in patients with post-reconstructive leg ocdema suggests increased capillary permeability to plasma proteins following vascular reconstruction.

The effect of brief illumination on intracellular free calcium concentration in cells with 5-aminolevulinic acid-induced protoporphyrin IX synthesis.

The effect of illumination on intracellular free calcium concentration, [Ca2+]i, was studied in a cell line (WiDr cells) derived from a primary adenocarcinoma of the rectosigmoid colon. In these cells the biosynthesis of protoporphyrin IX was stimulated by 5-aminolevulinic acid to reach levels of 600-700 pmol of protoporphyrin IX per mg cell protein. A brief (1-min) exposure of the cells to light (70% of light energy at 340-380 nm) resulted in an increase in [Ca2+]i. This increase was not reversible over a period of at least 20 min following illumination. Elevation of [Ca2+]i most probably represented an influx of calcium ions from the medium to the cell, since it was completely abolished in the presence of extracellular EGTA. The increased [Ca2+]i did not reflect general membrane damage, as determined by trypan blue staining as well as measurement of the intercalation of ethidium bromide into cellular DNA, and neither did the sustained elevation of [Ca2+]i lead to any substantial loss of clonogenicity following illumination of protoporphyrin-containing cells. Together these results indicate that an increased [Ca2+]i level is not per se a cause of cell death during photodynamic therapy.

Protein concentration of subcutaneous interstitial fluid in the human leg. A comparison between the wick technique and the blister suction technique.

The wick technique and the blister suction technique are the most common methods for sampling of subcutaneous interstitial tissue fluid in man. The blister suction technique has the advantage of being less invasive than the wick technique, but the reliability of this method is still controversial. The aim of this study was to evaluate whether the simpler blister suction technique using large (8 mm) blisters could replace the wick technique in the investigation of patients with postreconstructive leg edema. Fifteen patients with ipsilateral leg edema following infrainguinal bypass surgery for lower limb atherosclerosis were investigated. The two different fluid sampling techniques were applied simultaneously on both legs. The concentration of total protein and albumin as well as colloid osmotic pressure of the subcutaneous interstitial tissue fluid in the leg were measured in all fluid samples. Agreement analysis was applied to compare the two methods, while the correspondence between the methods was estimated with linear regression analysis. The agreement index was found to be positive for all variables from the operated as well as from the contralateral control limb. Furthermore, all values were within the agreement limit. The best agreement between the two methods was found for colloid osmotic pressure on the operated side. According to the equation of linear regression, there was a slight overestimation of the wick values compared to the observed blister values. In conclusion, there was a good methodological agreement between the blister suction technique and the wick technique. The less invasive blister suction technique should be regarded as the method of choice for the investigation of subcutaneous interstitial tissue fluid in patients with postreconstructive leg edema.

The interaction of gadolinium complexes with isolated rat hepatocytes.

The lanthanide metal, gadolinium, is currently used in contrast agents for magnetic resonance imaging. We have performed a study of the interaction between isolated rat hepatocytes and 153Gd complexed to diethylene-triamine pentaacetic acid (DTPA) or to DTPA-albumin conjugates. The study shows that isolated hepatocytes are able to take up both types of 153Gd complexes. The 153Gd-DTPA-albumin complexes are apparently taken up by pinocytosis, and possibly receptor-mediated endocytosis and/or adsorptive endocytosis, whereas the uptake mechanism of 153Gd-DTPA is unknown. The 153Gd-DTPA-albumin complexes, but not the 153Gd-DTPA complex, are degraded by the cell. The degradation is inhibited by ammonium chloride. Gadolinium is slowly released back to the medium after loading of the cells with both complex types. In the experiments reported here no evidence of any adverse effects on the hepatocyte resulting from exposure to the 153Gd-complexes were observed.

[The new medical curriculum in Trondheim. Organization and planning of a new curriculum]. / Det nye medisinstudiet i Trondheim. Organisering og planlegging av et nytt studium.

In autumn 1993, the Faculty of Medicine in Trondheim will be able to offer complete undergraduate medical education to 60 students per year, after 18 years of 3 1/2 years' education, based only on the clinical part of undergraduate medical school. The entire new curriculum is based on problem-based learning with the preclinical and the clinical part of the study totally integrated for 5 1/2 years. Throughout the curriculum any health care problem will be analyzed in terms of three perspectives: the biological, the environmental and the behavioural. The new medical curriculum was planned as a project during which both teachers and students took part in planning groups organized on several levels. Through clinical demonstrations and a course on the doctor-patient-relationship, the 60 students will meet patients as a natural part of their study from day one. In the Family Practice and in the Skills Lab they will acquire clinical skills during the first two years. In the fourth and fifth years the students spend two 8-week clerkship periods in community hospitals and community health practices.

Genetic carrier detection in Norwegian families with acute intermittent porphyria.

Early detection of carriers of acute intermittent porphyria (AIP) is of great value as an assistance for correct diagnosis and prevention of attacks. In order to complement traditional biochemical methods, restriction fragment length polymorphism (RFLP) studies as well as analysis for a previously identified point mutation were included in a study of three Norwegian AIP families. Several asymptomatic carriers could be identified, and the study thus demonstrates the usefulness of the combination of biochemical and genetic analysis.

The transferrin receptor: its diagnostic value and its potential as therapeutic target.

Transferrin receptors are present on almost all mammalian cells. The receptor participates in the cellular acquisition of iron from transferrin by receptor-mediated endocytosis. Receptor abundancy is generally regulated by two factors: i) cellular iron status and ii) cell growth. These two factors form the basis for the utilization of transferrin receptor determination as a diagnostic tool. In the assessment of body iron status and erythropoietic activity the measurement of circulating transferrin receptor has proved to be of value as a measure of mild tissue iron deficiency, to distinguish iron deficiency anemia from the anemias of chronic disease, and as a sensitive index of iron deficiency during pregnancy. Histochemical analysis of the presence and abundancy of the transferrin receptor will continue to serve as an additional tool in special cases to distinguish between malignant and normal cell growth, and to provide additional information about the biological behaviour of tumor cells. Finally, the transferrin receptor holds a potential as a target for direct and indirect drug delivery in the therapy of malignant cell growth.

[Quality assurance in laboratory medicine--reality and challenge]. / Kvalitetssikring i laboratoriemedisin--realiteter og utfordringer.

So far, quality assurance in laboratory medicine in Norway has concentrated mainly on results. As yet, however, much still remains to be done, particularly in the fields of pathology, radiology and nuclear medicine. Insufficient emphasis has been paid to quality assurance of the requests made for tests and interpretation of the results. Important for this part of quality assurance are teaching and education, cooperation between the laboratory physician and the clinician, more active participation of the laboratory physician in the solving of clinical problems and a change from menu-oriented to problem-oriented requests.

Subcellular localization of transferrin and pyrophosphate in liver cells after perfusion in situ or incubation in vitro.

Rat livers were perfused in situ with [125I]-transferrin, FITC-dextran and [32P]-inorganic pyrophosphate (PP). In a parallel set of experiments rat liver homogenates was incubated with [125I]-transferrin and [32P]-PPi. In both sets of experiments cellular organelles were prepared by density gradient centrifugation in metrizamide. In the perfusion experiments [125I]-transferrin and FITC-dextran accumulated in fractions rich in plasma membrane enzymes, whereas in the homogenate experiments [125I]-transferrin remained in soluble fractions. [32P]-PPi remained in soluble fractions in both sets of experiments, i.e. PPi did not co-localize with transferrin taken up by intact cells. In another set of experiments fractions rich in endosomes were tested for their ability to synthesize PPi from ATP. No PPi was found. The results argue against a role for PPi in transferrin-iron release within endosomes.

Variations in serum erythropoietin and transferrin receptor during phlebotomy therapy of hereditary hemochromatosis: a case report.

Serum levels of transferrin receptor and erythropoietin were determined in 2 patients with hereditary hemochromatosis undergoing phlebotomy therapy. The objective of the study was to determine changes in serum transferrin receptor and serum erythropoietin occurring during therapy, and to investigate if such changes could be useful to monitor the therapy. The study showed that serum transferrin receptor, and to a lesser extent serum erythropoietin, may be better parameters than serum ferritin as indicators of when phlebotomy should be discontinued. The most sensitive parameter, however, appeared to be the serum transferrin receptor/ferritin ratio.

[Laboratory diagnosis of multiple sclerosis]. / Laboratoriediagnostikk ved multippel skelerose.

In 26 patients with multiple sclerosis 100% responded abnormally to magnetic resonance imaging of the brain. Lesions in the posterior fossa were observed in 18 patients. The auditory brain stem response was abnormal in 15 patients, and 22 had abnormal immunoglobulins in the cerebrospinal fluid. The correlation between abnormalities of the auditory brain stem response and the magnetic resonance images was greatest in a subgroup where the two investigations were performed within a ten day interval. Results from magnetic resonance imaging, evoked potentials and cerebrospinal fluid investigations were used to reclassify 13 of 15 patients with clinically "possible" or "probable" multiple sclerosis to a higher level using Poser's criteria. Evoked potentials (the auditory brain stem response in particular) correlated best with clinical multiple sclerosis category. We recommend that magnetic resonance imaging is established as a first-hand investigation in evaluations of multiple sclerosis. Evoked potentials and cerebrospinal fluid investigations may prove to be more specific, however, and these investigations should also be performed as a routine.

Quality control does improve the performance of laboratory practice in primary health care.

Quality control surveys combined with teaching courses and expert consultations were offered to laboratories serving general practitioners in the county of Sør-Trøndelag, Norway. Fifty-one laboratories participated in the programme and the surveys during 1988-89. During the study period the number of laboratories having acceptable accuracy of their haemoglobin determination increased from approximately 80% to more than 90%. A similar increase was found also in precision where CV declined from 6.7% to approximately 2%. Additional positive effects were more close co-operation and more fruitful dialogues between the specialist laboratory and that of the general practitioners. The results offer promising perspectives to the control of laboratory performance in primary health care and the optimal use of laboratory tests.

[Clinical chemistry in Norway from the beginning of the 1990's]. / Klinisk kjemi i Norge ved inngangen til 1990-årene.

From 1980 to 1989 the number of chemical tests ordered in Norway increased by approximately 81%. By 1989 it amounted to approximately eight tests per inhabitant per year. The increase has been most marked among outpatients. During the period, the trend has been towards closer cooperation between the laboratory and the primary health care physician, and greater focus on rational use of laboratory resources. Challenges in the nineties are recruiting, to improve cooperation between the clinician and the laboratory, and to put more resources into research.

[Nikolai Astrup and his asthma]. / Nikolai Astrup og hans astma.

Nikolai Astrup (1880-1928) is recognized as one of the most famous Norwegian painters of his time. He grew up in Jllster, in the western part of Norway, and has become known as the painter of the West Coast, not only because of his landscapes but even more so because of the atmosphere and special mood reflected in his paintings. Most of his paintings depict scenes from spring and summer nights in Jllster. From a medical point of view it is interesting to know that Astrup suffered from asthma all his life, and one wonders whether his motives appeared to him during long walks at night while plagued by severe attacks of asthma. One also wonders what sort of impact the disease had on the composition of his paintings. There is reason to believe that his asthma not only hampered his creative activity and brought his life to a premature end, but also influenced his visionary imagination and helped him to discover the motives for his pictures.

Brain-stem auditory evoked potentials in multiple sclerosis: the relation to VEP, SEP and CSF immunoglobulins.

One hundred patients with multiple sclerosis (MS) were analysed retrospectively with respect to investigations of brain-stem auditory evoked potentials (BAEP), pattern reversal visual evoked potentials (VEP), somatosensory evoked potentials (SEP), and cerebrospinal fluid immunoglobulins (CSF-IG). BAEP were abnormal in 42% of those with normal VEP and SEP examinations, and in 38% of patients with normal CSF-IG. The chance of obtaining at least one abnormal EP was lower in patients with normal CSF-IG than in patients with abnormal CSF. When a "dispersion ratio" was included in the criteria for BAEP abnormality, the sensitivity increased compared with conventional BAEP criteria. We recommend that BAEP should still be included in the EP test battery for patients with suspected MS.