Karl Jacob

Capillary gas chromatographic injection system for large sample volumes

Abstract The injection system for capillary gas chromatography described can solve problems encountered in the trace analysis of high-boiling compounds. It increases the overall sensitivity, because the whole sample can be used for the gas chromatographic separation and detection. Up to 250 μl can be injected with an injection rate of about 1–10 μl/sec. The retention times are not affected by the volume injected or the nature of the solvents used. The linearity of the response is not influenced by the sample volume. The reporducibility (coefficient of variation) of the peak areas is less than 1%. The handling of the injector is simple and the glass insert can be changed in less than 1 min.

Induction of a chemoattractive proinflammatory cytokine response after stimulation of keratinocytes with Propionibacterium acnes and coproporphyrin III

Background  The inflammation in acne vulgaris is widely thought to be induced by an immunological reaction, but the role of Propionibacterium acnes is unclear.

Determination of serum cortisol by isotope-dilution liquid-chromatography electrospray ionization tandem mass spectrometry with on-line extraction.

Abstract A liquid chromatographic-mass spectrometric method for the determination of cortisol in serum using atmospheric pressure electrospray ionization and tandem mass spectrometry is described. During sample preparation, 150 μl of serum were deproteinized with methanol/zinc sulfate followed by on-line solid phase extraction employing column switching. Tri-deuterated cortisol was used as the internal standard. The following transitions were monitored: cortisol, 363>309 m/z; d3-cortisol, 366>312 m/z. The total run-time was 5 minutes. The method proved linear (0–500 μg/l; r=0.999), precise (total coefficient of variation between 5.0% and 3.2% at a mean cortisol concentration of 15.1 μg/l and 269 μg/l, respectively; n=16) and specific with regard to relevant endogenous and exogenous steroids.

Diagnosis of porphyrias by ion-pair high-performance liquid chromatography

Ion-pair reversed-phase high-performance liquid chromatography together with fluorescence detection is useful in the analysis of urinary porphyrin carboxylic acids. The sensitive and quantitative detection facilitates the clinical diagnosis of porphyrias. The method described permits the detection of porphyrins down to 0.1 ng directly from urine without laborious sample pre-treatment. A linear response curve was obtained from 0.2 up to 200 ng for coproporphyrin I. The within-assay correlation coefficients ranged from 2.5 to 10.1%. Recovery experiments gave an accuracy of 89-109%. The rapidity and simplicity of the method allows its application to the routine analysis of urinary porphyrins in the clinical laboratory.

Corticosteroid-binding globulin and free cortisol in the early postoperative period after cardiac surgery.

OBJECTIVES To characterize concentrations of corticosteroid-binding globulin (CBG), total and free serum cortisol, and free urinary cortisol in patients during the postoperative period of cardiac surgery. DESIGN AND METHODS In 24 patients serum was sampled on the first and second postoperative day after cardiac surgery (21 procedures with thoracotomy, 3 thoracoscopic procedures); urine was collected for two 10-h periods (8 P.M. until 6 A.M.) on the respective postoperative days. Total serum cortisol and free urinary cortisol were measured with an automated chemiluminescence assay (analysis of urine after extraction with dichloromethane), and CBG using a coated-tube RIA. Free serum cortisol was calculated from the concentrations of total serum cortisol and CBG as described previously. Thirty healthy volunteers were studied as controls. RESULTS CBG was reduced to about one-half of the normal concentration on both postoperative days. Whereas total cortisol was about two-fold increased on the first postoperative day compared to controls extremely high concentrations of free serum cortisol were calculated from CBG and total cortisol [median 136 nmol/L (interquartile range 100-185); controls 21.8 nmol/L (interquartile range 16.9-29.8)]. On the second postoperative day, median total serum cortisol was within the interquartile range of the controls, free serum cortisol in contrast was still two-fold increased. Free serum cortisol and free urinary cortisol were significantly correlated (r = 0.60). CONCLUSIONS Extremely high concentrations of free serum cortisol are typically found in the postoperative period of cardiac surgery; under these conditions the mere consideration of total cortisol does not appropriately display the activation of the adrenal cortex.

In vivo porphyrin production by P. acnes in untreated acne patients and its modulation by acne treatment

Propionibacterium acnes is often discussed as a contributing pathogenic factor in the aetiology of acne lesions. The aim of this study was to test which porphyrin patterns are synthesized by P. acnes in vivo in untreated acne patients and during standard acne regimens. These photosensitive compounds are potential targets for photo-dynamic therapy of acne and need to be better characterized in the skin. Using high-performance liquid chromatography coproporphyrin III was the main porphyrin identified in all patients. Coproporphyrin I and protoporphyrin were found at considerably lower concentrations. When the porphyrin concentration of individual patients receiving isotretinoin was analysed repeatedly over time, clinical improvement was associated with lowered levels of porphyrins. Statistical analysis demonstrated a significant reduction in the porphyrin fractions only in the isotretinoin group which was associated with clinical improvement 2 months after starting therapy.

Derivatization method for the high-sensitive determination of amines and amino acids as dimethylthiophosphinic amides with the alkali flame-ionization detector

Abstract A derivatization method for the high-sensitive gas chromatographic determination of primary amines with teh alkali flame-ionization detector is presented Amino-containing compounds easily react with dimethylthiophosphinic chloride in the presence of triethylamine between −20 and +20°. These derivatives show good gas chromatographic properties. The detection limit of N-dimethylthiophosphinylaniline was 500 fg. The mass spectra of these amine derivatives were studied. The fragmentation pathway were elucidated by means of precise mass measurements and decoupled metastable transition determinations.

Serum cortisol/cortisone ratio after Synacthen stimulation

OBJECTIVES 11beta-Hydroxysteroid dehydrogenase (11beta-HSD) enzymes interconvert active cortisol and inactive cortisone. There is growing evidence that local tissue concentrations of cortisol are generally modulated by site specific different 11beta-HSD actions. While 11beta-HSD type 2 unidirectionally inactivates cortisol, type 1 isoform acts bidirectionally. 11beta-HSD type 1 is mainly localized in the liver and may thus restore circulating biologically inactive cortisone to active cortisol thereby modulating systemic glucocorticoid action; such a mechanism might be of importance in stressful situations. To study this hypothesis we investigated the influence of exogenous ACTH on serum cortisol/cortisone ratio. DESIGN AND METHODS Paired serum samples that were submitted for routine analysis of cortisol before and 1 h after stimulation with 250 microg ACTH (1-24) (Synacthen) were collected prospectively if the routine tests indicated normal adrenal function; 40 patients were included in the study, 29 patients were female, 11 male, median age was 31 yr (range 14-70). Serum cortisol and cortisone were determined using LC-ESI/MS/MS with an online sample extraction system and tri-deuterated cortisol as the internal standard. RESULTS While mean serum cortisol increased by 109% (mean basal concentration 373 nmol/L (SD 151 nmol/L), stimulated 781 nmol/L (SD 194 nmol/L)), mean serum cortisone significantly decreased after ACTH administration by 31% (p < 0.001, paired t-test for differences). Mean serum cortisone was 70 nmol/L (SD 16 nmol/L) before and 48 nmol (SD 16 nmol/L) after ACTH administration; decrease in serum cortisone was observed in 34 (85%) of the patients. The ratio of serum cortisol/cortisone increased in all subjects (mean 5.4 (SD 1.9) before ACTH, and 16.2 (SD 6.2) after ACTH; p < 0.001). CONCLUSIONS The data of our observational study suggest a modulation of peripheral metabolism of cortisol by ACTH with a stimulation of systemic 11beta-HSD type 1 activity, leading to restoration of inactive cortisone to biologically active cortisol.

Improved separation and detection of free porphyrins by high-performance liquid chromatography

Porphyrins were separated using ion-pair reversed-phase high-performance liquid chromatography. The eluents were aqueous potassium phosphate buffer and tetrabutylammonium phosphate in methanol. The influences of pH value and ionic strength of the phosphate buffer and molarity of the ion-pair reagent in methanol were investigated to improve separation and detection. A linear response curve was obtained from 0.38 to 7.64 pmol for coproporphyrin I. The detection limits were determined to be 0.12 pmol for coproporphyrin I and 0.22 pmol for uroporphyrin I.

Interdependence between degree of porphyrin excess and disease severity in congenital erythropoietic porphyria (Günther's disease)

Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance in autosomal recessive congenital erythropoietic porphyria (Günther’s disease). Although the clinical manifestations have been well described since Günther’s fundamental observations, an interdependence between disease severity and porphyrin excess has yet to be elucidated. We investigated porphyrin metabolism in nine Indian patients suffering from the characteristic clinical symptoms: skin photosensitivity, red-colored urine as a sign of extremely elevated porphyrinuria and mild to severe hemolytic anemia. Porphyrins in urine, feces and blood were analysed by HPTLC and HPLC in conjunction with spectrophotometry and spectrofluorometry. Uroporphyrinogen III synthase activities in red blood cells were determined using a coupled-enzyme assay. Biochemical studies revealed varying degrees of porphyrinuria with total urinary porphyrins between 23 and 102 μmol/24 h (normal <0.2 μmol/24 h) and uroporphyrin predominance. Urinary and fecal coproporphyrin isomer I were markedly elevated to 87– 97% and 81–93% (normal <31%, <75%), respectively. Overproduction of porphyrins led to a considerable porphyrinemia with mainly copro- and protoporphyrin. A hitherto undescribed fecal porphyrin pattern with increased protoporphyrin levels was found in three patients. This atypical finding was probably related to severe hemolysis since protoporphyrin can be excreted only via the liver with bile in the feces. High porphyrin levels in urine, feces and blood were associated with worse cutaneous symptoms. Activities of uroporphyrinogen III synthase in red blood cell lysates were decreased to between 9% and 30% of controls. Patients showed increased porphobilinogen deaminase activities, up to 190% of control. Deficiency of uroporphyrinogen III synthase activity was reflected by inversion of the relationship between and isomer III leading to dominance of isomer I. Elevation of porphobilinogen deaminase activities is related to hemolysis and, additionally, to regulatory compensation for the enzyme deficiency. Variations in both the severity of photosensitivity and the enhancement of porphyrin production and excretion indicate the molecular heterogeneity of this disease. These findings suggest a close relationship between the metabolic disturbance reflected by porphyrin excess and the severity of disease expression.