Enzymatic Determination of Lactose in Ortho Tri-Cyclen

Lactose is a very important sugar because of its abundance in the milk of humans and domestic animals (2). It is a valuable asset as a basic nutrient and the main substrate in fermentative processes that led to the production of fermented milk products, such as yogurt and kefir (2). Lactose is also an inactive ingredient in prescriptive medications. For example, Othro Tri-Cyclen is an oral contraceptive that contains an unknown amount and concentration of lactose. Lactose is believed to be located in tablets that do not contain the active hormonal ingredients. Each white tablet contains 0.180 mg of the progestational compound, norgestimate (18,19-Dinor-17-pregn-4- en-20-yn-3-one,17-( acetyloxy)-13-ethyl-, oxime,(17?)-(+)-), 0.035 mg of the estrogenic compound, ethinyl estradiol (19-nor-17?-pregna, 1,3,5( 10)-trien-20-yne-3,17-diol), lactose, magnesium stearate, and pregelatinized starch. Each light blue tablet contains 0.215 mg of the progestational compound norgestimate (18,19-Dinor-17-pregn-4- en-20-yn-3-one, 17-( acetyloxy)-13-ethyl-, oxime,( 17?)-(+)-), 0.035 mg of the estrogenic compound, ethinyl estradiol (19-nor-17?-pregna, 1,3,5 (10)-trien-20-yne-3,17-diol), FD&C Blue No.2 Aluminum Lake, lactose, magnesium stearate, and pregelatinized starch.

Although the quantities of active ingredients are listed, the quantity and even concentration of lactose and some other inactive ingredients are not. This poses the question of what the concentration of lactose is in the Ortho Tri-Cyclen. Is the amount per tablet enough to irritate individuals with extreme lactose intolerance? Instructions for daily usage encourage users to take two tablets within one day if the individual forgets to take the tablet from the day before. Consquently, if the concentration of lactose is doubled, is this enough to irritate an individual with lactose intolerance? This experiment attempts to measure the concentration of lactose within four tablets in Ortho Tri-Cyclen - and three "active pills" and one "sugar" pill. The "sugar" pill is green and the active pills vary in color from dark blue, to light blue, to white. The results of this experiment are compared to the given values for lactose intolerant levels and foods with hidden lactose are also considered (6).

There are many methods within the field of chemistry that are used to analyze lactose such as Multi-enzyme biosensors with amperometric detection and enzymatic splitting of disaccharides. In a mutli-enzyme biosenor method, the lactose is decomposed and oxidized by the immobilized enzymes and the hydrogen peroxide generated during the enzymatic reactions is determined by amperometric detection (3). In the enzyme splitting method, disaccharides are split by enzymes and specific measurements are taken of the resulting glucose by a Beckman Glucose-Analyzer (4). However, due to limited time and resources, more common and simple methods were researched for this experiment. Popular and relatively inexpensive methods for determining the concentration of lactose are enzymatic methods that use spectrophotometry and beta-galactosidases. This is in part due to the idea that lactose-hydrolyzing enzymes are structurally and phylogenetically related to different types of beta-galactosidases and bacterial cellobiases involved in the enzymatic degradation of cellulose (2). The method used in this experiment is similar to one used for powdered milk due to the powder-like composition the tablet can be crushed into and treated as if it were a powdered milk.

In the presence of ß-galactosidase and water, lactose is hydrolyzed to D-glucose and D-galactose. In the presence of Gal-DH, D-galactose is oxidized to galactonic acid by NAD+. The NADH formed is stoichiometrically dependent on the amounts of lactose and is quantitatively determined by measuring the absorbance of the solution at

340 nm (5):

Lactose + H2O ß-galactosidase ? D-glucose + D-galactose

[1]
D-galactose + NAD+ Gal-DH ? galactonic acid + NADH[2]
The product, NADH, is stoichiometrically dependent on the amounts of lactose and galactose present and can be therefore measured by absorbance (1).

Apparatus and Supplies

Micropipettes 20.00, 50.00, 100.00 and 200.00 uL

pH meter

Spectrophotometer

1 cm light path cuvettes to measure at 340 nm

Magnesium sulfate - 625.00 mg

Trisodium citrate dihydrate - 2.8 g

Sulfuric acid - 2.00 mol/L

Sodium Hydroxide - 0.1 mol/L and 1 mol/L

Nicotinamide adenine dinucleotide - 25 mg

Potassium dihydrogen phosphate - 8.3 g

ß-galactosidase - 5.00 mg/mL
Gal-DH - 5.00 mg/mL
Potassium hexacyanoferrate hydrate - 3.60 g

Zinc sulfate hydrate - 7.20 g

10.00 mL glass bottles

Method

Reagent and Buffer

A 0.2 M of citrate buffer at pH 6.6 was prepared. Trisodium citrate dihydrate (2.8 g), 42.00 mg of citric acid monohydrate, and 625.00 of mg magnesium sulfate heptahydrate were dissolved in 40.00 mL DI water. Sulfuric acid and sodium hydroxide were used to adjust the pH to 6.6. The solution was diluted with 50.00 mL of DI water. The buffer was stored at 4ºC. NAD+- citrate buffer was prepared. NAD+ (25.00 mg) was prepared in 5.00 mL citrate buffer and stored at 4ºC. The phosphate buffer was prepared at pH 8.6. Potassium dihydrogen phosphate (8.3 g) was dissolved in 40.00 mL of DI water and diluted with 50.00 mL of DI water. The solution was stored at 4ºC. ß-galactosidase (5.00 mg/mL undiluted) and 5.00 mg/mL of galactose dehydrgenase
(Gal-DH undiluted) were used.

Sample

The mass was obtained for the white, light blue, dark blue, and green tablets. The tablets (complete pill per bottle) were placed into 10.00 mL of distilled water. The samples were diluted again in another 10.00 mL of distilled water. Each sample was filtered with a syringe and transferred to 10.00 mL bottles.

Determination

The reagents were used at room temperature (20ºC-25ºC) and the total volume of the sample, enzyme, and DI water was 3.32 mL. Table 1 and Table 2 were used for the quantity of reagents, sample, buffer, and water that needed to be transferred into the cuvettes for absorbance measurements. After the respective amounts of each reagent in Table 1 are added, the solution was mixed and incubated in the cuvettes at room temperature for 15 minutes. An initial absorbance measurement was taken and 0.05 mL of Gal-DH was added to each cuvette by careful mixing with the pipette tip. For the first trial, the machine was set to take readings so that a time for when the activity stopped could be estimated. The activity did not cease after 45 minutes. Each reaction of the calibration curve and of the samples was stopped at 20 minutes thereafter to decrease lab time and create a standard set for all of the data. All samples were ran only twice due to lack of remaining enzyme for triplicate analysis. The differences in absorbance from initial to final absorbance readings were determined (Figure 1). This procedure was conducted using a standard curve with the concentration range of 1.6 x 10-5 to 1.6 x 10-2 in which was calculated from the protocol of Mustratanta & Ostman (1).

Calculation

To calculate lactose concentration, a standard curve (Table 3) was prepared and graphed using lactose concentration by total absorbance (Figure 1 and Figure 2) in Microsoft Excel. The total absorbance of the samples were then compared to the standard curve through the equation:

y = 41.599x + 0.9553

[3]
An ANOVA statistical test was performed using SPSS (Table 5) to accept or reject the null hypothesis of lactose concentration being higher in the "sugar" pill than the other pills.

Results and Discussion

The standard curve resulted in total absorbance measurements from 0.748 to 1.594 with concentration increasing respectively. The reaction seems to have become saturated towards the last absorbance measurement of the standard curve; therefore, the last point on the graph was taken off and only the linear range was plotted (Figure 2). The samples were ran in the same fashion that the standard curve was in which each sample had a 20-minute reaction time. This is because the enzymatic reaction would not stop after 45 minutes. The second absorbance measurement of the white pill was taken out for analysis purposes and only the first measurement for the white pill was used for the calculations. Therefore, there was no standard deviation or RSD calculated for the white pill (Table 4).
The light blue had the highest lactose content with 0.0115 grams and the white pill had the lowest content. The green pill is the "sugar" pill and was expected to have the highest concentration because it did not contain any estrogenic compounds like the other color pills do. However, after an ANOVA statistical test, the probability of my results, assuming the null hypothesis, is 0.211 (Table 5). The F score is way above 0.05 cut off, so it cannot be concluded that the pills are statistically significantly different from one another.

None of the numerical values of lactose in any of the pills was comparable to the levels need to induce lactose intolerance. Even with the addition of lactose in hidden foods such as powdered meals and supplements, the amount is still not enough to cause harm due to the lactose in the pill having such a low concentration. When the number of grams in each pill is double or tripled, the amount still falls short of the 12-18 grams (Table 6) need to induce a reaction. It is safe to assume that Ortho Tri-Cyclen will not cause lactose intolerance reactions when ingested at regular and high dosages.

Some sources of error that can account for the results is that the enzyme used in this experiment was not the same grade as the enzyme used in Mustratanta & Ostman's. This could also be the reason as to why the Gal-DH in this experiment reacted for 45 minutes without signs of stopping. Another source of error is the calibration curve. A concentration range had to be calculated from scratch and was not given in the Mustratanta & Ostman protocol. This could be a source of error because the birth control pills contain smaller concentrations of lactose than the food analyzed by Mustratanta & Ostman. Both enzymes were placed into a beaker with ice to keep cool. This may have interfered with the effectiveness of the enzymes because they were allowed to incubate in the beaker for over eight hours when they should have been in the freezer at 4°C.

If the experiment were to be replicated in the future, there are several changes that are suggested. One suggestion is to lower concentrations of lactose for the standard curve. Another suggestion is to try different brands of birth control pills including the morning after pill. These other brands of birth control such as Yasmin and Modicon contain different amounts of estrogenic compounds and therefore may contain different amounts of lactose as well. The results can be compared to the results of Ortho Tri-Cyclen. A third suggestion would be to order a different brand or grade of enzyme. The results may vary for different enzymes prepared by different companies.

References
(1) Mustratanta, A.; Ostman, C. Biotechnology and Food Research. 1997, 80, 584-590.
(2) Adam, A. C.; Rubio-Texeira, M.; Polaina, J. Food Science & Nutrition. 2004, 44, 553-557.
(3) Adányi, N; Szabó, E. E.; Váradi, M. European Food Research and Technology. 1999, 209, 220 - 226.
(4) Dörner, K.M. European Journal of Pediatrics. 1977, 126, 45 - 52
(5) Methods of Biochemical Analysis and Food Analysis. 1989, 70, 80-82.
(6) Swagerty, D.L. ; Walling, A.D.; Klein, R. M. American Family Physician, 2001, 65, 60.