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Acetic acid (acetate) is converted to acetyl-CoA in the presence of the enzyme acetyl-CoA synthetase (ACS) by adenosine-5'-triphosphate (ATP) and coenzyme A (CoA).

\(\text{(1) Acetate + ATP + CoA }\space ^{\underrightarrow{\text{ACS}}} \space \space\text{Acetyl-CoA + AMP + Pyrophosphate}\)

Acetyl-CoA reacts with oxaloacetate in the presence of citrate synthase (CS) to form citrate.

\(\text{(2) Acetyl-CoA + Oxalacetate + H}_2\text{O }\space ^{\underrightarrow{\text{CS}}} \space \space\text{Citrate + CoA}\)

The oxaloacetic acid required for reaction (2) is produced from malic acid and nicotinamide adenine dinucleotide (NAD) in the presence of malate dehydrogenase (MDH). In doing so, NAD is reduced to NADH:

\(\text{(3) L-Malate + NAD}^+ \space ^{\underrightarrow{\text{L-MDH}}} \space \text{Oxalacetate + NADH + H}^+\)

The formation of NADH+H⁺ forms the basis of this analysis, which is measured as an increase in the absorbance at 340 nm. Since this concerns a previous indicator reaction, the quantity of NADH+H⁺ is not linearly proportional to the acetic acid concentration.

Glucose and fructose are phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P) and fructose 6-phosphate (F-6-P):

\(\text{Glucose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{G-6-P + ADP}\)

\(\text{Fructose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{F-6-P + ADP}\)

In the presence of the enzyme glucose-6-phosphate dehydrogenase (G6P-DH), G-6-P is oxidized from nicotinamide adenine dinucleotide phosphate (NADP⁺) to gluconate-6-phosphate. Reduced nicotinamide adenine dinucleotide phosphate (NADP + H⁺) is formed:

\(\text{G-6-P + NADP}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{Gluconate-6-phosphate + NADPH + H}^+\)

The amount of NADP + H⁺ formed during the reaction is equivalent to the amount of glucose. NADPH + H⁺ is a measurand and is determined based on its absorbance at 340 nm.

After the reaction is complete, F-6-P is converted to G-6-P by phosphoglucose isomerase (PGI):

\(\text{F-6-P} \space ^{\underrightarrow{\text{PGI}}} \space \text{G-6-P}\)

The amount of NADPH + H⁺ formed during the reaction is equivalent to the amount of fructose. NADPH + H⁺ is the parameter being measured and is determined on the basis of its absorption at 340 nm.

G-6-P reacts in turn with NADP⁺ to form gluconate-6-phosphate and NADP + H⁺. The additional amount of NADP + H⁺ formed is equivalent to the amount of fructose and is determined photometrically based on its absorption at 340 nm.

Note:

Alternatively, NAD⁺/NAD + H⁺ can be used instead of NADP⁺/NADPH + H⁺:

\(\text{G-6-P + NAD}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{Gluconate-6-Phosphate + NAD + H}^+\)

Titratable acidity represents the sum of the free acids present in a beverage, with the exception of the dissolved carbon dioxide (carbonic acid). In fruit juices and the beverages prepared from them, they usually consist of malic acid, citric acid and tartaric acid.

The titration of the degassed beverage sample (freed from carbonic acid) is carried out potentiometrically using 0.25 mol/l sodium hydroxide solution either to a pH of 7.0 calculated as tartaric acid or to a pH of 8.1 calculated as citric acid.

Formic acid can be found in beer in small quantities, and it is also formed by contaminating bacteria, for example, by lactic acid bacteria (rods).

In the presence of the enzyme formate dehydrogenase (FDH), the formic acid created in this reaction is quantitatively oxidized to bicarbonate by nicotinamide adenine dinucleotide (NAD):

\(\text{Formic acide (HCOO}^- \text{) + NAD}^+ \space\text{+ H}{_2}\text{O }\space ^{\underrightarrow{\text{FDH}}} \space \text{CO}{_2}\text{ + NADH + H}^+\)

The amount of NADH+H⁺ formed during the reaction is equivalent to the amount of formic acid and can be determined photometrically based on its absorbance at 340 nm.

L-Malic acid (L-malate) is oxidized by nicotinamide adenine dinucleotide (NAD) in the presence of L-malate dehydrogenase (L-MDH) to oxaloacetic acid:

\(\text{L-Malic acid + NAD}^+ \space ^{\underrightarrow{\text{L-MDH}}} \space \text{Oxaloacetic acid}\text{ + NADH + H}^+\)

The equilibrium of this reaction disproportionately favors malic acid. However, the oxaloacetic acid can be captured with the help of a downstream reaction involving the enzyme glutamate-oxaloacetate-transaminase (GOT) in the presence of L-glutamic acid, shifting the reaction in favor of oxaloacetic acid and NADH + H+:

\(\text{Oxaloacetic acid + L-Glutamate} \space ^{\underrightarrow{\text{GOT}}} \space \text{L-Aspartate + 2-Oxoglutarate}\)

The amount of NADH+H⁺ produced during the reaction is equivalent to the quantity of L-malic acid and its absorption is determined photometrically at 340 nm.

Glucose is phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P).

\(\text{Glucose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{G-6-P + ADP}\)

In the presence of the enzyme glucose-6-phosphate dehydrogenase (G6P-DH), G-6-P is oxidized by nicotinamide adenine dinucleotide phosphate (NADP⁺) to gluconate-6-phosphate. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is formed:

\(\text{G-6-P + NADP}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{gluconate-6-phosphate + NADPH + H}^+\)

The amount of NADPH+H⁺ formed during the reaction is equivalent to the amount of glucose. NADPH+H⁺ is determined based upon its absorbance at 340 nm.

Note:

Alternatively,  NAD⁺/NADH + H⁺ can be used instead of NADP⁺/NADPH+ H⁺:

\(\text{G-6-P + NAD}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{Gluconate-6-Phosphate + NAD + H}^+\)