Hydroxyproline assay

Hydroxyproline assay. Hydroxyproline was determined according to Jamall et al. (1981). The assay employs p-dimethylaminobenzaldehyde (Ehrlich s reagent), which forms colored products with pyrroles originating from hydroxyproline oxidation. The values thus determined for hydroxyproline mass were multiplied by 8.0 to obtain the corresponding collagen mass. 

It is the purpose of this review to outline critically the methods available for hydroxyproline assay, and to summarize the information obtained by hydroxyproline determinations in urine, plasma (or serum), and the tissues, with emphasis on understanding the physiological and pathological setting in which these measurements have been made. No attempt will be made to discuss recent developments in the chemistry (H3, H4) or the biosynthesis (Ul) of collagen, since thorough reviews of these topics are available. 

Many of the chromatographic procedures are applicable to the measurement of labeled hydroxyproline in general they do not allow for multiple simultaneous determinations. Since collagen hydroxyproline is labeled only by administering its precursor, proline, in labeled form, the difficulties in radioactive studies have arisen largely because the nonhydroxy-proline metabolic products of proline or proline itself have not been rigorously separated from hydroxyproline or hydroxyproline-derived pyrrole. This is the purpose of the more recently introduced modifications for labeled hydroxyproline assay (Jll, L12). It is too early to know whether one or both of these will suffice for general use. 

A new approach to hydroxyproline assay is a recently introduced enzymatic procedure based on the ability of a multiple-enzyme system in adapted Pseudomonas strains of bacteria to degrade hydroxyproline (R7). Also, a standard colorimetric procedure has been adapted for use in an AutoAnalyzer apparatus (G5). Automatic amino acid analyzers can be easily programmed to isolate hydroxyproline from its more usual place in the shoulder of the aspartic acid peak (see M15). 

L12. LeRoy, E. C., Harris, E. D., and Sjoerdsma, A., A modified procedure for radioactive hydroxyproline assay in urine and tissues after labeled proline administration. Anal. Biochem. 17, 377-382 (1966). 

Several adequate methods for hydroxyproline are available. Generally, a method for detection of protein-bound hydroxyproline is required, inasmuch as collagenases break down collagen into peptides and not free amino acids. Hydroxyproline assays are useful on many occasions, such as providing quantitative determinations of the degree of lysis of collagen gel plates. 

Samples were taken from each solution after incubation for calcium determination. Samples of the collagenase incubations were assayed for hydroxyproline. 

The reduction was done essentially as described previously (Robins, 1976). After several washings with 0.9% (w/v) sodium chloride, 0.05 M sodium phosphate, pH 7.4, at 4°C, the stirred demineralized root powder was reduced with 0.71 g sodium borohydride, an estimated one-twentieth of the collagen mass. After one hour, the solution was acidified (pH < 4, pH paper) to inactivate remaining borohydride. Thereafter, the pH was readjusted to 7.5. The solution was centrifuged and the pellets were hydrolyzed by heating for 24 hours at 112°C in 6 M HCl under N2 in bottles with Teflon-sided screw caps. The dark hydrolyzate was paper-filtered, evaporated under reduced pressure at 50°C, and mixed with 5 ml 0.1 M acetic acid. The hydrolyzate was assayed for hydroxyproline. 

Special assays (urine and plasma) lactate, orotidine, thiosulphate, carnitine, succinylacetone, hydroxyproline, urate, orotate, sialic acid, MPS, guanidino-acetate, HVA, pyroglutamate, 5H1AA, pipecolate, pyruvate, 3-hydroxybutyrate, phytanate, VLCFA, homocysteine, 7-dehydrocholesterol, phenylalanine ERNDIM 8 148... 

Identification Pork Collagen contains not less than 5.52% hydroxyproline, corresponding to 40.0% collagen, as determined under Assay (below). 

Kivirikko KI, Laitinen O, Prockop DJ. Modification of a specific assay for hydroxyproline in urine. Anal Biochem 1967 19 249-55. 

Fw. 1. Series of reactions involved in the colorimetric assay of hydroxyproline, demonstrating the oxidation and decarboxylation of hydroxyproline to pyrrol (S14). 

Since the presence of hydroxyprohne is unique in collagen elastin contains a small amount), the determination of collagen content in a coUagen-rich tissue is readily done by assaying the hydroxyproline content. 

The reaction is the basis of various methods for the determination of amino acids since it is possible to measure (1) the CO2 produced, (2) the NH3 produced and (3) the colour intensity obtained when the liberated ammonia reacts with a further molecule of ninhydrin to produce a purple compound which can be assayed photometrically. This is the method by which amino acids are estimated using an amino acid analyser. The imino acids (proline and hydroxyproline) give yellow products instead of a purple one. Amino acids give a strong reaction with ninhydrin, but proteins and polypeptides, which contain far fewer free amino groups, give a much weaker reaction. 

When Severo Ochoa left the Biochemistry Department at New York University I had to search for another mentor and I was fortunate to be able to continue my graduate work with Albert Keston. While developing the isotope derivative method for amino acid assay we showed for the first time that hydroxyproline was uniquely present in collagen. This observation made a great impression on me. Furthermore, I had the good fortune while at New York University Medical School to meet and to get to know the late Joseph Bunim. It was from him that I learned about the role of collagen in connective tissue and also about collagen diseases. 

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