Hydroxyproline and hydroxylysine in different tissues of human embryos.

The collagen content in various tissues of human embryos was studied at four different steps of the maturation process. The umbilical cord of a 16-17 week old embryo was found to be active in collagen biosynthesis, Tibia, articular cartilage and skin showed of a peak of total hydroxyproline in the 16-17th week, decreasing later on, while the hydroxyproline decreased in umbilical cord from the 15-16th week to the 24th week. Hydroxylysine followed the hydroxyproline changes in articular cartilage and umbilical cord.

Liquid chromatography/mass spectrometry methods for distinguishing N-oxides from hydroxylated compounds

This study describes the application of liquid chromatography/mass spectrometry (LC/MS) methods for distinguishing between aliphatic and aromatic hydroxylations and between hydroxylations and N-oxidations. Hydroxylations and N-oxidations are common biotransformation reactions of drugs. Electrospray (ESI) and atmospheric pressure chemical ionization (APCI) were used to generate ions from liquid chromatographic effluents. ESI-MS, ESI-MS/MS, APCI-MS, and APCI-MS/MS experiments were performed on several metabolites and derivatives of loratadine (a long-acting and nonsedating tricyclic antihistamine) using an ion trap mass spectrometer (LCQ) and a triple-quadrupole mass spectrometer (TSQ). The observations are as follows: (1) LC/ESI-MS produced predominantly [M + H]+ ions with minor fragmentation. (2) LC/ESI-MS/MS data, however, showed a predominant loss of water from metabolites with aliphatic hydroxylation while the loss of water was not favored when hydroxylation was phenolic. N-Oxides (aromatic and aliphatic) showed only a small amount of water loss in the MS/MS spectra. (3) Under LC/APCI-MS conditions, aliphatic hydroxylation could be readily distinguished from aromatic hydroxylation based on the extent of water loss. In addition, N-oxides produced distinct [M + H - O]+ ions. These [M + H - O]+ ions were not produced in the APCI-MS spectra of hydroxylated metabolites. (4) Similar to the ESI-MS/MS spectra, the APCI-MS/MS spectra from the (M + H)+ ions of N-oxides yielded a small amount of water loss but no [M + H - O]+ ions. These results indicate that LC/APCI-MS can be used to distinguish between hydroxylated metabolites and N-oxides.

Automated triple assay for proline, hydroxyproline and hydroxylysine on one single sample.

1. A new, quick, sensitive and specific assay for the quantitative determination of proline is presented together with its automatization. The use of glacial acetic acid-formaldehyde as the solvent for ninhydrin lends a series of advantages to the reaction. 2. A serial automated procedure for the simultaneous detection of proline, hydroxyproline and hydroxylysine in one single sample is pesented. The advantage of the triple assay lies in the use of one single sample instead of three previously aliquoted fractions in three individual automated assays. The triple assay is as specific, reproducible and sensitive as the individual assays. 3. A quick, manual assay for hydroxylysine is presented. This method represents a further simplifiction of previous ones by the same author and adds the advantage of rapidity to their sensitivity and specificity.

Three automated quantitative assays for serum proteins.

Three quantitative assays for detection of proteins are reported. One is an adaptation to the Auto-Analyzer of the widely used method of Lowry et al. In another procedure, the product of the reaction of proteins with a modified biuret reacts with the phosphomolybdic--phosphotungstic reagent of Folin and Ciocalteu. The third method, performed on hydrolyzed sample, is based on the measurement of proline (Pro) and the conversion of the Pro content into protein. The universal presence of proline in serum proteins suggested this assay. The values for total serum proteins as assayed by the three procedures are similar. The second and the third assay for proteins, described in this paper, can also be performed manually. Studies on interfering substances and their elimination as well as on the sensitivity of the assays are reported.

An assay for total hexosamine and a differential assay for glucosamine and galactosamine.

Two new procedures are presented for quantitative determination of glucosamine and galactosamine. One, which is proposed for total hexosamine, yields chromogens of equal intensity with equal concentration of glucosamine and galactosamine. There is addition of the correspondent chromogens when they are present in mixtures. The procedure is presented as a manual as well as an automated assay. The other procedure is a differential assay which allows the detection of galactosamine without interference by glucosamine. By the two procedures, the hexosamines present in acid mucopolysaccharides and/or glycoproteins can be determined.

Micromethod for fractionation of acid mucopolysaccharides.

A new micromethod for fractionation of acid mucopolysaccharides based upon the use of different concentrations of HC1 to separate the complex of CPC with non-sulphated, monosulphated and polysulphated acid mucopolysaccharides (glycosaminoglycans) is presented. The method utilizes the different binding of the anionic macromolecules to the cationic compound cetyl pyridinium chloride. The method is simple and reproducible. The use of HC1 as eluent allows the exclusion of some steps required when salts are used for elution. Hexuronic acids are determined on the eluents.

Simplified method for determination of protocollagen proline hydroxylase.

Preparation of (14C) Pro labeled protocollagen and requirements for its hydroxylation by PPH was investigated. Protocollagen can be prepared by centrifugation of the biological material at 15.000 X g. Substances in current use, viz catalase, bovine serum albumin and dithiothreitol were found unnecessary under the hydroxylation conditions used. A decrease in PPH in the testis and skin of rats with increasing age was demonstrated.

An LC/MS/MS method for the quantitation of MTIC (5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide), a bioconversion product of temozolomide, in rat and dog plasma.

A sensitive and selective HPLC/electrospray ionization tandem mass spectrometric (LC/ESI/MS/MS) method for the quantitative determination of MTIC (5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide), a pharmacologically active hydrolysis product of temozolomide, was developed and validated over a linear range from 10 to 400 ng ml(-1) in dog plasma and from 10 to 500 ng ml(-1) in rat plasma. This HPLC method utilized small plasma volumes (70 microl), rapid sample processing, and isocratic elusion conditions to achieve sensitive and selective MS/MS detection. Samples were processed and analyzed one at a time every 4.5 min in order to compensate for the inherent instability of MTIC. Both MTIC and the internal standard DTIC [5-(3,3'-N,N'-dimethyltriazen-1-yl)-imidazole-4-carboxamide] were quantitated in the positive ion, selected reaction monitoring (SRM) mode. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in the plasma from both species. Inter-assay accuracy and precision of all calibration standards and quality control (QC) samples were within +/- 11 and 12%, respectively, with the exception of the LLOQ in rat plasma (17%). The validated method was used to determine the time dependent plasma concentration of MTIC in rats and dogs following a single oral dose of temozolomide. The standard curve and the quality control data indicate that the method performed acceptably throughout the sample analysis period.