THEBIOMEDICAL SCIENCE SCIENCE THEBIOMEDICAL 26 SCIENTIST The big story The big story SCIENTIST 27 CLINICAL CHEMISTRY CLASSICSPT 3 In the third of our series looking at landmark moments Fig. 1. Molecular model of the enzyme alpha-amylase in clinical chemistry, Stephen Clarke turns his Fig. 2. French chemist Anselme Payen attention to amylase and alkaline phosphatase. Fig. 3. The US biochemist Michael Somogyi 1 rom the vast methodology evolutionary genetic studies have Amylase is now known to be a dependent developed by the US surgeon Robert papers in clinical obtained and results literature in clinical chemistry, demonstrated their ancient history.(1) metalloenzyme, which catalyses the Elman (1897-1956) who promoted the chemistry, notably calculated from time this third short review selects Diastase, later known as amylase, was the hydrolysis of links between adjacent value of serum amylase in acute on blood protein taken into Somogyi two landmark papers with first enzyme to be discovered by Payen glucose units to break down complex pancreatitis with studies between 1927 precipitants, methods units with a reference background notes, paying and Peroz in 1833, when it was shown carbohydrates, such as starch to dextrins, and 1937.(6) He later became world for blood sugar and range of 80-180 tribute to those early pioneers that alcoholic extracts of malt could maltotriose, maltose and glucose. renowned for supplementary amino acid ketone bodies. units/100ml serum. 3 who developed long-serving convert starch to sugars.(2) It it was later Wohlgemuth used this amyloclastic nutrition, saving lives of war-scarred This landmark Modifications were assays for the analysis of blood shown that amylase was present in saliva (ALC) principle based on the decrease of victims of the Second World War. paper described made by Street and serum amylase and alkaline phosphatase (1845) and that pancreatic juice could starch-iodine colour after a standard modifications of both Close (1956) and by Ffor clinical purposes. Advances in protein digest starch (1846). period of incubation and many variations Michael Somogyi ALC and SCM methods Somogyi himself in 1960.(8) chemistry and a greater understanding of In 1908, amylase was the first were introduced, notably in the The Hungarian-born US biochemist realising the clinical needs for Chromogen-linked substrates enzyme catalysis and kinetics provided a enzyme to be quantitatively composition of starch and Michael Somogyi(7) worked at Washington an urgent simple, rapid, sensitive became popular during the 1970s foundation for enzyme assays, increasing assayed, by Julius endpoint determination. University School of Medicine in St Louis, and reliable procedure with the former notably “Phadebas” launched in 1970 the range of analytical approaches to the Wohlgemuth, at the Alternative assays were Missouri. With Edward Doisy and Philip and a more robust assay with the latter. until superseded by automated amylase investigation of clinical disease. University of Berlin, saccharometric (SCM), Shaffer, he prepared purified insulin for This popular ALC procedure used 1ml systems using well-defined substrates with a method which measuring the its first successful use in treatment in serum with a carefully prepared clear with auxiliary and indicator enzymes Blood serum amylase could be applied to increase in sugar after 1922. He moved to the Jewish hospital of corn starch preparation and standardised and NAD/NADH spectrophotometric Enzymes as biological catalysts are urine, blood or starch incubation,(5) or St Louis in 1926 for his remaining iodine with timed withdraw until the red end points or substrates releasing 4 (3-4) 2 (9) essential to life processes and duodenal fluid. viscosimetric, notably LIBRARY PHOTO DESIGN/SCIENCE FOUNDATION/LAGUNA HERITAGE ©CHEMICAL IMAGES: professional career and published over 70 brown colour of erthyrodextrin was nitrophenol for colorimetric assays. THEBIOMEDICAL SCIENCE 28 SCIENTIST The big story The flurry of scientific research Fig. 4. British biochemist Sir Robert Robison and development Blood serum alkaline the Banting Institute at the University in 1930 to 1960 phosphatase of Toronto as Head of Pathology and Studies of the hydrolysis of phosphoesters reported this paper with his colleague, has borne fruit were made by Suzuki (1907), and Grosser A Riley Armstrong. This method with the (1912) found this reaction occurred in alternative ‘Bodansky’ procedure became many tissues.(10) Significant advances in the substantive manual methods for understanding bone mineralisation were around 30 years prior to automation. King made by the British biochemist, Robert and Amstrong preferred sodium phenyl Robison at the Lister Institute – he phosphate as substrate more rapidly isolated active alkaline phosphatase (ALP) hydrolysed by ALP reducing the from animal bone extracts in 1923 used as incubation time and sample volume. a biochemical reagent to explore possible The substrate was buffered to pH9 substrates.(11) In 1924 Robison and Herbert and phenol measured colorimetrically Kay showed that phosphoesters were with Folin and Ciocalteu reagent present in blood plasma and in 1929 Kay (phosphotungstic and phosphomolybdic devised a method for plasma ALP using acids) to produce a blue chromogen read incubation with beta glycerophosphate at 640nm against a phenol standard. (BGP) and measurement of phosphate Enzyme activity was recorded in KA units phenols in the presence of an alkaline- produced and found greatly raised levels defined as the liberation of 1mg of phenol reducing agent, potassium ferricyanide, in certain active bone diseases.(12) In 1930 per 100ml serum with a reference range to produce a red quinol for a more rapid Roberts demonstrated raised results in of 3-13KA units. Greatly raised results are and stable colour(16), this was later adapted obstructive jaundice and both Kay and seen in bone disease, such as rickets and for automation. It had been long known Roberts later improved their methods. osteomalacia and liver diseases, with that ALP was present in tissues and Russian-born US biochemist Aaron increases in infective hepatitis and more isoenzymes have been detected in kidney, Bodansky favoured BGP procedure and in so in obstructive jaundice. intestinal mucosa and placenta using 1933, using a more sensitive phosphate King and colleagues at Hammersmith electrophoretic techniques(17) or by reagent, was able to reduce incubation developed a method for combining serum resistance to heat, liver isoenzyme is heat time and sample volume and his method phosphate and ALP determinations(15) and stable, while bone isoenzyme is heat was commonly used in the USA. Both Kay later devised a procedure for ALP which labile. Structural studies show that and Bodansky defined their unit of used amino-antipyrine to react with human ALP is a zinc-containing activity measurement and reference metalloenzyme with some structural ranges. The value of ALP in bone and liver differences between isoenzymes and diseases became the main focus. four structural genes encoding for ALP have been cloned, sequenced and mapped EJ King and AR Armstrong to chromosomes.(18) Earl Judson King (1901-1962) was a Amylase and alkaline phosphatase Canadian-born biochemist who was a measurements have proved to be leading force in the development of extremely valuable aids in diagnostic clinical biochemistry in England while and therapeutic medicine. The flurry Head of Pathology at the Postgraduate of scientific research and development Medical School in Hammersmith and between 1930 and 1960 has borne fruit later as Professor of Biochemistry at the and became the foundation of clinical University of London. He is noted for his enzymology inspired by those, such as emphasis on practical methods suitable Michael Somogyi and Earl King and for even small laboratories and for the use many others who have been all too briefly of micro methods. (13-14) mentioned in this short review. He developed a lifelong interest in organic phosphates and related enzymes Stephen Clarke is a retired IBMS fellow. while spending a period with Robison in He previously worked in clinical 4 IMAGE: SCIENCE PHOTO LIBRARY PHOTO SCIENCE IMAGE: 1928. He returned to Canada working at chemistry at Southmead Hospital, Bristol..