Description of Instrumentation and Procedures - Advances in Biochemical Engineering Biotechnology - page 18

Typical sample matrices are blood, serum and fermentation broth. Oxalate in
urine was also measured using oxalate oxidase [24]. Substrate recycling offered
routes for highly sensitive measurements. An example was lactate (or pyruvate)
determination with a co-immobilized LDH/LOD (lactate dehydrogenase/lactate
oxidase) column that repeatedly oxidizes lactate to pyruvate and reduces pyru-
vate to lactate. Each cycle produces a considerable amount of heat [15]. A simi-
lar approach was employed for the determination of NAD + /NADH by co-enzy-
me recycling by using lactate dehydrogenase plus glucose-6-phosphate dehy-
drogenase, and of ATP/ADP by coupling pyruvate kinase with hexokinase as the
recycling enzymes. By operating at excessive glucose supply, a hexokinase
column was used for indirect assay of ATP with micromolar sensitivity.A multi-
plicative effect could be attained by coupling the recycling systems of pyruvate
kinase and of LDH/LOD [25].
Monitoring of specific proteins eluted from chromatographic columns was
demonstrated using the ET as a direct online monitor for purification of pro-
teins/enzymes. As an example, LDH was recovered from a solution by affinity
binding of N 6 -(6-aminohexyl)-AMP-Sepharose gel, and the signal from the ET
was used to regulate the addition of the AMP-Sepharose suspension to the LDH
solution [26, 27].

3.2
Industrial and Process Monitoring

For bioprocess monitoring, the ET was employed in the assay of penicillin in
fermentation broth, using -lactamase or penicillin acylase [28]. Also, ethanol

generated in alcohol fermentation by yeast was monitored using alcohol oxida-
se. Other metabolites that were monitored during fermentations include lactate,
glycerol, acetaldehyde, sucrose and glutamine [29].

b

3.3
Clinical Applications

Metabolites in human blood are closely associated with the state of an individ-
ual's health. Determination of metabolites is critical in clinical diagnosis, since
they can serve as a criterion for judging the severity of the sickness. Of these
metabolites, glucose, lactate and urea are most frequently determined. Special
attention to determination of glucose in blood is due to the fact that diabetes is
well known as a dangerous and widespread disease that results in a high glucose
concentration in the blood. Some other effects of metabolites, such as urea and
lactate, on shock, respiratory insufficiency, and heart and kidney diseases are
understood to some extent.

3.3.1
In-Vitro Monitoring

For determination of the metabolites in human blood, samples were collected
from veins into heparinized or EDTA containing tubes. For glucose analysis, NaF