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doi: 10.1002/bmc.1130010302pmid: 3506825
In the clinical laboratory, paper chromatography is still the most useful, simple, inexpensive procedure for initial identification of abnormalities of amino acid excretion. The results of its use for more than 8000 paediatric and adult renal patients is surveyed. Nonspecific generalized aminoaciduria was the most frequent abnormality found, comprising some 70% of abnormal results, with cystine‐lysinuria the next most common. The identification of the abnormal excretory pattern of amino acids as distinct from the normal was complicated by the effects of the New Zealand diet. In particular, valine, citrulline, hydroxyproline and glutamic acid are found in considerable amounts as part of the normal pattern. Their dietary origin is discussed. Varying mixtures of monosaccharides and disaccharides occurred in association with a range of amino acid patterns.
Margison, Jennifer M.; Wilkinson, P. M.; Cerny, T.; Thatcher, N.
doi: 10.1002/bmc.1130010303pmid: 3506819
A high performance liquid chromatography method is described for measuring Ifosfamide (I) in human serum. This involves solvent extraction, reverse phase HPLC and UV detection at 190 nm. Standard curves of peak height × detector sensitivity versus I concentration in serum were linear with a lower limit of detection of 100 ng/ml. Authentic 14C‐labelled I cochromatographed with standard I and with I found in serum from treated patients. The concentration‐time curves of I determined by both HPLC and gas chromatography were indistinguishable. We conclude that this method is suitable for determining I pharmacokinetics in biological specimens.
Koenderman, Anky H. L.; Loppen, Piet L.; Marinus, Lian A. M.; van den Eijnden, Dirk H.
doi: 10.1002/bmc.1130010304pmid: 2976281
A sensitive HPLC method for the assay of UDP‐GlcNAc:β‐galactoside β1 → 3‐N‐acetylglucosaminyltransferase activity was developed. Using lactose as an acceptor, the formation of the product GlcNAcβ1 → 3Galβ1 → 4Glc can be determined without interference by substrates resulting from enzymatic and chemical breakdown of the donor substrate UDP‐GlcNAc. The method is very specific since products of other transferase reactions, which potentially may be formed in the incubations in vitro, elute at positions different from that of GlcNAcβ1 → 3Galβ1 → 4Glc. By use of this assay method it could be demonstrated that normal and malignant hematopoietic cells and cell‐lines, with the exception of erythrocytes and reticulocytes, contain β1 → 3‐N‐acetylglucosaminyltransferase activity.
Morris, George S.; Simmonds, H. Anne; Davies, Phillip M.
doi: 10.1002/bmc.1130010305pmid: 3506820
Inherited purine and pyrimidine disorders may be associated with serious, sometimes life‐threatening consequences. Early and accurate diagnosis is essential. Difficulties encountered when using existing high pressure liquid chromatographic (HPLC) methods led to the development of an improved method based on prior fractionation of urine. The advantages are as follows. 1. Production of fingerprints demonstrating altered urinary excretion patterns characteristic of any one of ten different disorders, in 30 minutes. 2. Positive identification and quantification by comparison with established methods (using conventional chromatography, electrophoresis and UV spectrophotometry) in addition to specific retention times and characteristic UV absorbance ratios at two separate wavelengths (245 and 280 nm) by HPLC. 3. Direct analysis of all the purines and pyrimidines normally found in human body fluids as well as identification of abnormal compounds. 4. Short time between successive analyses while maintaining excellent resolution between compounds of interest and column longevity. 5. Improved separation of the different adenine‐based compounds encountered in some disorders, plus demonstration of potential interference by dietary or drug metabolites. 6. Applicability to the monitoring of therapy involving a variety of different purine and pyrimidine analogues. Particular attention should be paid to sample preparation. Plasma profiles will confirm the diagnosis in some, but not all, of these disorders.
Hsi, Kuo‐ling; Wu, Shih‐xiang; Chen, Zhong‐guang; Guo, Xiu‐yue; Tsou, Kang
doi: 10.1002/bmc.1130010306pmid: 3506821
A biologically active peptide designated hLCP has been isolated and purified to homogeneity from human lung carcinoma by means of acidic extraction and successive chromatography on Sephadex G‐50, Toyopearl HW‐40 F and reverse‐phase high performance liquid chromatography columns. Analysis showed that peptide consists of thirteen amino acids. Primary structure of hLCP has been deduced by double‐coupling Edman degradation combined with enzyme digestion as H‐Ser‐Pro‐Pro‐Asp‐Gly‐Lys‐Lys‐Glx‐Ser‐Ala‐Asp‐Val‐Lys‐OH. hLCP possesed significant excitatory activity on an electrical stimulation induced contraction. No hLCP could be detected in normal lung tissue. The possibility of using hLCP as a biochemical marker in the clinic for the early detection of lung carcinoma is being investigated.
doi: 10.1002/bmc.1130010307pmid: 3506822
Metabolites of 2,4,6‐trinitrotoluene (TNT) were found in the urine of a group of TNT munition workers. The urine extracts were analysed by micro liquid chromatography/mass spectrometry. The metabolites found included 2‐amino‐4,6‐dinitrotoluene, 4‐amino‐2,6‐dinitrotoluene, 2,4‐diamino‐6‐nitrotoluene, 2,6‐diamino‐4‐nitrotoluene and untransformed TNT. The detection limit of the metabolites in urine was 0.1 ng/ml for 20 ml urine samples.
Takeshita, Hisayoshi; Desiderio, Dominic M.; Fridland, Genevieve
doi: 10.1002/bmc.1130010308pmid: 3506823
A combination of gradient reversed‐phase high performance liquid chromatography (RP/HPLC) with a radioreceptor assay detector that uses two ligands is used to obtain effectively the metabolic profile of endogenous receptoractive opioid peptides in the canine pituitary and in seven selected brain regions including the hypothalamus, caudate nucleus, mid‐brain, amygdala, thalamus, pons‐medulla, and the hippocampus. Gradient RP/HPLC separates a mixture of endogenous peptides over a wide range of hydrophobicities. A novel opioid preparation from canine limbic system synaptosomes is utilized in a radioreceptorassay screen; tritiated etorphine (ET) or D‐2ala, D‐5leu‐leucine enkephalin (DADL) is used as the competitively displaced ligand. This receptor‐rich preparation contains several receptor types, and thus serves well as a screen with the required low level of specificity. Subsequent analysis with other detectors of high specificity (MS, RIA) will follow this screen in other studies. Etorphine interacts with several of the opioid peptide‐preferring receptors, whereas DADL is more specific towards the delta receptor that preferentially binds the smaller pentapeptides of the enkephalin family. The highest amount of peptide receptor activity found in this study is in the pituitary tissue, a smaller amount in the hypothalamus and caudate nucleus, and still lower amounts in the other five brain tissue extracts. This variation in peptide concentration most probably reflects three separate factors that operate in this biologic system: differential tissue‐specific processing patterns of the large peptide precursors; distribution of the three opioid peptide systems; and the receptor preparation and the radioligand used in the assay. The structures of the receptoractive compounds in each RP/HPLC peak await mass spectrometric confirmation.
Bhamra, R. K.; Flanagan, R. J.; Holt, D. W.
doi: 10.1002/bmc.1130010309pmid: 3506824
A simple HPLC method for penbutolol and 4‐hydroxypenbutolol assay has been developed. Plasma or serum (200 μl) is vortex‐mixed (30 s) with Tris solution (2 M, pH 10.6) containing an internal standard (50 μl) and methyl t‐butyl ether (200 μl). After centrifugation, the extract (100 μl) is analysed using an unmodified silica column (250 × 5 mm ID) and iso‐octane–methanol–methyl t‐butyl ether (55:25:20) containing ammonium perchlorate (10 mM, pH 5.7) as eluent and with fluorescence detection. No interference has been encountered and the limit of accurate measurement for both compounds is 5 μg/l.
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