Human Seminal Plasma Fructose Determination Method Validated

Human semen is the secretion of the male reproductive organs, containing sperm cells and seminal plasma (SP), a complex mixture of testicular, prostatic, and accessory gland secretion that provides biochemical support for ejaculate function.

Assessing the quality of human semen and its fertilizing capacity therefore requires not only qualitative and quantitative analysis of spermatozoa, but also biochemical analyses of the SP. A rapid, simple, specific, and quantitative seminal fructose photometric determination by hexokinase and phosphoglucose-isomerase using a semi-automated bichromatic analyzer has been developed.


Medical biochemists at the Merkur University Hospital (Zagreb, Croatia) used leftover semen samples from males undergoing routine fertility evaluation whose ages ranged from 28 to 41 years, with normal semen parameters in their study. No commercial quality control material was available, so two in-house pools of SP were used for internal quality control, yielding coefficients of variation of 0.85% and 1.06% for normal and pathological values, respectively. The aim of this study was to validate the performance of the enzymatic method for SP fructose on a Beckman Coulter (BC) AU400 analyzer (Olympus Mishima, Sunto-gun, Japan), as well as to challenge the performance of the method regarding quality.

The determination of SP fructose by the enzymatic method is carried out indirectly via glucose. After fructose phosphorylation with hexokinase, the resulting fructose 6-phosphate is converted into glucose 6-phosphate by phosphoglucose isomerase. Glucose 6-phosphate is further oxidized by glucose 6-phosphate dehydrogenase, yielding 6-phosphogluconate and Nicotinamide adenine dinucleotide (NADH). The amount of NADH formed is stoichiometric with the amount of D-fructose and D-glucose, so the measured rate of NADH formation reflects quantitatively the amount of glucose and is equivalent to the amount of SP fructose.

The methods initially compared 43 SP samples across the linearity range of 0.37 to 24.7 mmol/L. Fructose was also measured in 20 SP samples obtained from healthy volunteers without fertility problems, to confirm the previously established reference intervals of 8.3 to 27.8 mmol/L. All measured samples were within the reference interval and the results were 8.8 to 23.6 mmol/L.

The authors concluded that the results of this verification study showed that the automatic enzymatic determination of SP fructose on a BC AU400 analyzer met the desirable quality specifications and requirements for the accreditation according to the ISO 15189 Standard, thereby providing a reliable diagnostic method for the clinical assessment of male infertility. The study was published in the July 2016 issue of the Journal of Applied Laboratory Medicine.

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