The differentiation of the porphyrias by means of high pressure liquid chromatography

The analysis of faecal and urinary porphyrins by high pressure liquid chromatography (H.P.L.C.) provides characteristic profiles and facilitates rapid diagnosis of variegate (porphyria cutanea tarda hereditaria), symptomatic porphyria (porphyria cutanea tarda symptomatica), hereditary coproporphyria, acute intermittent porphyria, erythro-hepatic protoporphyria and congenital porphyria (erythropoietic porphyria).     

A modification of the pisano method for vanilmandelic acid using high pressure liquid chromatography

A method for the measurement of urinary vanilmandelic acid (VMA) is described based on a modification of the colorimetric procedure of Pisano. VMA is oxidized to vanillin with subsequent measurement of the vanillin by high pressure liquid chromatography with electrochemical detection (LCEC). The technique has been successfully applied to a number of human and animal urine samples. Urinary VMA values obtained by the LCEC method are in good agreement with those determined by the method of Pisano. The new method is both more selective and more sensitive than the colorimetric procedure.     

Determination of deuterium-labeled phenylalanine and tyrosine in human plasma with high pressure liquid chromatography and mass spectrometry.

A method is presented for the recovery of deuterated phenylalanine and tyrosine from human plasma. Phenylthiohydantoine derivatives are formed (Edman reaction) which are separated and isolated by high pressure liquid chromatography. The relative concentration of the deuterated amino acid is determined by mass spectrometry. The results obtained from a healthy person after oral loading with 40% monodeuterated L-phenylalanine are presented. The method appears to be suitable for in vivo studies of phenylalanine metabolism in humans.     

Hypoxanthine-guanine phosphoribosyl transferase: assay using high performance liquid chromatography

A method is described for the estimation of hypoxanthine-guanine phosphoribosyltransferase using high performance liquid chromatography.The inosine monophosphate (IMP) generated from hypoxanthine is determined, after separation on a C₁₈ reversed phase silica column with a buffer-methanol gradient, by the absorbance at 254 nm. Simultaneous reciprocal measurement of hypoxanthine consumption is made. The assay is suitable for screening red cell lysates for hypoxanthine-guanine phosphoribosyltransferase deficiency; the results being expressed as nmol inosine monophosphate · h^?1 · mg^?1 haemoglobin.The normal range found was 94 ± 15 nmol IMP-h^?1 · mg^?1 haemoglobin and hypoxanthine-guanine phosphoribosyltransferase activities down to 1% of normal can be assayed accurately.     

The rapid determination of erythrocyte porphyrins using reversed-phase high performance liquid chromatography

The extraction, separation and quantification of free acid erythrocyte porphyrins are described. The porphyrins are extracted from whole blood using 3:1 ethyl acetate/acetic acid (v/v) and separated using the reversed-phase mode of high performance liquid chromatography (RPLC). The compounds are detected on-line spectrofluorometrically using an excitation wavelength of 404 nm and an emission cut-off filter of 550 nm. Excellent resolution of the porphyrin free acids is achieved in < 6 min. The analytical recoveries for protoporphyrin IX and zinc protoporphyrin IX were > 90% with relative standard deviations for day-to-day analysis under 6%.     

Analysis of lidocaine and its dealkylated metabolites by high-pressure liquid chromatography.

A high-pressure liquid chromatographic method is presented for determining lidocaine and its dealkylated metabolites, monoethylglycinexylidide and glycinexylidide, in plasma. This method uses 20-microliter samples injected directly through a pre-column onto a C18 analytical column. It is fast, sensitive and linear over a concentration range of 1--10 microgram/ml. Since the method requires no preliminary treatment of the plasma, it provides a practical means of monitoring lidocaine and its metabolites in therapeutic situations.     

Analysis of total urinary catecholamines by liquid chromatography: methodology, routine experience and clinical interpretations of results

A simple routine method is described for simultaneous assay of total urinary adrenaline, noradrenaline and dopamine. The catecholamines are pre-purified on a small ion-exchange column, separated by reversed phase ion-pair liquid chromatography, and are quantitated by electrochemical detection. The method was routinely applied to 422 urines. Elevated values were found in four urine specimens obtained from patients with histologically proven phaeochromocytomas. Virtually no interference by endogenous or exogenous compounds was found. Values for urinary catecholamines determined by fluorimetric analysis agreed with those obtained by high pressure liquid chromatography with electrochemical detection. Within-day CVs for the compounds ranged from 5.2-11.9%, between-day CVs from 3.3-6.6%. The normal range (95% confidence level) was 20-230 micrograms/24 h for noradrenaline and 1-35 micrograms/24 h for adrenaline.     

Determination of glycosylated adult and foetal haemoglobins by affinity chromatography

Estimation of adult glycosylated haemoglobin by affinity chromatography was found to be quick and less dependent on ionic strength, pH and temperature than ion-exchange chromatography. Results obtained by both procedures correlated strongly (r = 0.96) but the range for normal subjects was smaller with the affinity assay. The affinity method correlated equally well with the colorimetric assay (r = 0.95). However, the method did not measure all the glycosylated forms, and only half of the glycosylated species isolated by ion-exchange chromatography was bound to the affinity resin. It also showed that the amount of glycosylated haemoglobin in cord blood is less than in adult blood.     

Endogenous levels of free and conjugated urinary 3-methoxy-4-hydroxyphenylethyleneglycol in control subjects and patients with pheochromocytoma determined by reversed-phase liquid chromatography with electrochemical detection

A sensitive and specific reversed-phase liquid-chromatography (HPLC) method for the determination of urinary free and conjugated 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) was developed. Sample preparation is minimal and the method is ideally suited for routine clinical assays of this catecholamine metabolite.Reported in this paper are excretion profiles of control subjects and patients with pheochromocytoma. Quantitative results were expressed as μg MHPG per mg creatinine, in order to eliminate variations associated with the 24-h urine collection. The levels of free and conjugated MHPG, determined in 20 control subjects ranged between 0.04–0.11 μg/mg of creatinine and 0.48–1.25 μg/mg of creatinine, respectively. The corresponding levels of free and conjugated MHPG in 8 patients with pheochromocytoma were 0.29–1.21μg/mg of creatinine and 2.47–15.98 μg/mg of creatinine, respectively.The described liquid-chromatographic analysis, coupled with the highly sensitive electrochemical detection, provides a simple and reliable method for metabolic profiling of catecholamine by-products at their endogenous levels. This offers an attractive possibility for the detection of neural crest lesions which may cause problems in diagnosis because of their small size, infrequent occurrence and symptoms similar to those of essential hypertension.     

The estimation of plasma valproate by gas-liquid chromatography.

A gas chromatographic method for the plasma assay of the anticonvulsant, sodium valproate, is described. Derivatization is not necessary. 200 microliter plasma are required for a single estimation. The method involves a chloroform extraction of valproate and the internal standard, cyclopentane carboxylic acid, from acidified plasma. Gas-liquid chromatography using the stationary phase 10% SP-216-PS gives complete separation of valproate and the internal standard in eight minutes. The limit of detection is 20 mumol valproate/1 plasma (equivalent to 40 pmol on column). This is well below the lower therapeutic plasma level. The between-run precision of the method indicates a variation for each sample within+/-3% of its mean value.     

Determination of 3-mercaptolactate, mercaptoacetate and N-acetylcysteine in urine by gas chromatography

A method is described for the determination of 3-mercaptolactate, mercaptoacetate (thioglycolate) and N-acetylcysteine in urine. As these compounds are mainly excreted as their mixed disulfides with cysteine, they are first reduced to the free thiols by an insoluble polymer containing thiol groups. After purification by chromatography on an organomercurial adsorbent, the compounds are converted to benzyl derivatives by extractive alkylation and determined by gas chromatography. The identity of the compounds analyzed was verified by mass spectrometry. It was demonstrated that mercaptolactate and mercaptoacetate are almost entirely excreted as their mixed disulfides with cysteine, whereas appreciable amounts of N-acetylcyteine are present as the symmetrical disulfide and the free thiol. The urinary excretion of the compounds from healthy human beings was also studied.     

Determination of bile acids in serum by capillary gas-liquid chromatography.

A glass capillary column and an appropriate relatively simple procedure for sample preparation have been developed for determination of serum bile acids. Sample preparation involved extraction with Amberlite XAD-2, solvolysis of sulfates, enzymatic hydrolysis with cholylglycine hydrolase, methylation and silylation. Because of complete chromatographic separation of bile acid trimethylsilylether derivatives from cholesterol on the capillary column, an additional step for elimination of cholesterol could be omitted. Trimethylsilylether derivatives were separated on a 20 meter x 0.3 mm i.d. glass capillary column covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20,000 as liquid phase according to Grob, K. and Grob, G. (1976) J. Chromatogr.125, 471--485, and Grob, K., Grob, G. and Grob, Jr., K., (1977) Chromatographia 10, 181--187. Overall recovery of the major human conjugated bile acids ranged from 86 to 89%. Reproducibility of bile acid determination was satisfactory in both normal and pathological serum with elevated bile acid concentrations (coefficient of variation 7.6 to 10.0%). The mean concentrations of cholic, deoxycholic, chenodeoxycholic and lithocholic acid in the serum of healthy subjects were 0.9, 1.0, 1.7 and 0.2 mumol/l in males, and 1.0, 0.8, 1.4 and 0.2 mumol/l in females.     

Separation and quantification of urinary di- and polyamines by gas chromatography with electron capture detection

A sensitive and specific gas chromatographic assay procedure employing electron capture detection has been developed for the assay of free and total di- and polyamines in human urine. Urine samples, hydrolysed with hydrochloric acid where necessary for the measurement of total amine output, were evaporated to dryness and, after the residues had been taken up in water, purified successively on Porapak Q and Dowex 50 X2 columns. Following evaporation of eluate, pentafluoropropionyl derivatives were made and analysed gas chromatographically using temperature programming. Di- and polyamines can be measured accurately at the picomole level and normal urinary output values calculated using this method agree well with those noted by other workers.     

Specific quantitation of plasma medroxyprogesterone acetate by gas chromatography/mass spectrometry

Investigation of various derivatives (O-methyloxime, trifluoroenol acetate and t-butyldimethylsilyl enol ether) coupled with the efficient preparation of a trideuterated analogue of medroxyprogesterone acetate, has allowed the development of a rapid and accurate assay for its quantitation in plasma by isotope dilution gas chromatography/mass spectrometry. High oral doses (2.8 g weekly) of medroxyprogesterone acetate are shown to lead consistently to plasma levels of less than 16 ng.ml-1.     

Plasma lipid analysis by thin-layer chromatography with flame ionisation detection and quantitation.

A method is presented whereby cholesterol, cholesterol ester, triacylglycerol, non-esterified fatty acid and polar lipid in plasma may be simultaneously quantitated. Following the addition of umbelliferyl palmitate as internal standard, plasma (0.1 ml) is extracted with chloroform/methanol (2 : 1, v/v). A portion of the extract is chromatographed on silica gel fused to a narrow cylindrical quartz rod. After chromatography the rod is traversed through a flame ionisation detector and the responses recorded and integrated. The procedure is shown to be sensitive, linear, reproducible and to compare well with conventional methods for the determination of plasma lipid classes.     

Quantitative analysis of serum lipoproteins by micro-scale thin-layer chromatography.

A method has been devised for the complete chemical analysis of serum lipoproteins, in which the constituents are separated by thin-layer chromatography and then measured by means of a flame ionisation detector. Since the response of the detector differs for each constituent, it is necessary to use a previously prepared calibration curve for each one. A complete analysis can be obtained from a single run on about 20 microgram of lipoprotein. However, from 5--10 chromatograms are needed for an adequate degree of precision. The method, which could be adapted to the measurement of tissue lipids, takes less than 2 h to complete. This speed and simplicity seem to give the method considerable potential for the investigation of patients with disorders of lipid transport.     

Isolation of thyroxine-binding globulin (TBG) by immunoadsorption chromatography: some physical and immunochemical characteristics

Thyroxine-binding globulin (TBG) was isolated from pooled whole human serum by diethylaminoethyl (DEAE) Sephadex anion exchange chromatography followed by immunoadsorption chromatography on a cyanogen bromide-activated Sepharose 4B-sheep anti-human TBG immunoadsorbent. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) of the purified TBG revealed a major protein band with a molecular mass of 65000 and a weak band of molecular mass 54000 in both reducing and non-reducing buffers. Sedimentation velocity analysis revealed an S_20,w coefficient of 4.5S and a calculated molecular mass of 60000. Immunochemical analysis confirmed the purity of the TBG preparation which gave a single precipitin peak on two-dimensional immunoelectrophoresis.     

A patient with purine nucleoside phosphorylase deficiency: enzymological and metabolic aspects.

1. Enzymological and metabolic data in a patient with nucleoside phosphorylase (NP) deficiency are described. 2. Incubation of intact NP-deficient red cells with [14C]adenosine showed a rapid uptake and conversion to inosine. Almost no radioactivity was incorporated in the adenosine nucleotides and no hypoxanthine labeling could be detected. 3. Incubation with [14C]inosine resulted in a rapid conversion to IMP in the normal intact red cells but in an accumulation of inosine in the medium with the erythrocytes of the patient, proving again that a NP deficiency is present. 4. The high PRPP level found may result from impaired consumption due to lack of substrates for the salvage enzyme HGPRT. 5. Incubation with [14C]hypoxanthine and [14C]adenine showed that normal HGPRT and APRT activities were present in the NP-deficient red cells. 6. In serum and urine of the patient the levels of inosine and guanosine were considerably increased, while the serum and urinary levels of uric acid were very low. In the two deceased sisters NP deficiency was also strongly suggested by analyses of the serum purines, of stored deep frozen samples.     

HPLC analysis of aromatic amino acids,nucleosides, and bases in plasma of acute lymphocytic leukemics on chemotherapy

Plasma chromatograms — obtained by the reversed-phase mode of high performance liquid chromatography (HPLC) — of 19 subjects with acute lymphocytic leukemia (ALL) were compared to those of 19 normal individuals. ALL patients were in remission and on a methotrexate and 6-mercaptopurine maintenance regimen. The concentrations of the aromatic amino acids l-phenylalanine and l-tyro-sine, the nucleosides uridine, adenosine, inosine, and guanosine, as well as the bases hypoxanthine and xanthine, were elevated in the leukemic in comparison to the normal chromatograms. Highest inosine levels corresponded to leukemic subjects whose condition severely deteriorated with time. Patients with lower inosine levels are still in continuous remission.