A nitrogen balance experiment using simulated urine samples

Authors


Abstract

We describe an undergraduate laboratory experiment that combines the advantages of problem-based learning with the need for biochemistry students to become proficient in practical laboratory skills. It also avoids the need to obtain ethical approval for recruiting volunteers and eliminates any possible biosafety issues with the handling and disposal of large amounts of urine. Simulated human urine samples are prepared that contain urea, uric acid, and creatinine at concentrations that represent the levels expected in 2 liters of urine collected over 24 h from subjects on various protein diets or during different physiological states. The students measure the nitrogen-containing compounds in the “urine samples” using specific colorimetric assays and use the data they generate to derive knowledge about nitrogen balance and the excretory metabolism of amino acids.

The introduction of problem-based learning into many biochemistry courses has added an important means of encouraging active higher level learning [1]. However it has often meant that the wet-lab, hands-on, practical component of biochemistry is neglected. Many courses that cater to students in the health related professions do not include practical laboratory sessions at all and have chosen to replace them with paper-based exercises or problems. When ready-made data are provided for these problems, students lose the appreciation of how the data are actually obtained and of course become less proficient in standard laboratory skills. An important goal of biochemical training is to learn how to pose questions, plan and carry out experiments, and obtain, analyze, and interpret the data. For this reason we have deliberately maintained practical laboratory sessions for all of our courses, although the time spent on doing practical exercises has been reduced in favor of developing project-style sessions, which allow the students more time for planning, thinking, and discussion.

We teach a one-semester course on human metabolism. Typically the students who enroll for this course are in their second year of a science degree program, majoring in biochemistry, nutrition, or physiology, or are in their second year of an undergraduate professional degree in medicine, dentistry, pharmacy or medical laboratory technology, in which biochemistry is a requirement. All these students have completed one-semester introductory courses in general biology and chemistry as well as a foundational course in biochemistry, which covers the structure and function of proteins and outlines the main catabolic pathways of carbohydrates and lipids. The students become familiar with the vocabulary and basic concepts of these sciences but get only limited laboratory experience.

The practical sessions of the human metabolism course focus on aspects of the effects of exercise and diet on human subjects, usually student volunteers, and require the collection of blood and urine samples to analyze for metabolites and proteins. With the increase in awareness of the potential hazards of handling such samples our university, rightly and in keeping with national guidelines, now enforces strict biosafety requirements. These can be relatively easily achieved for small volume samples such as fingerprick blood samples, but for large volumes of urine collected from multiple student volunteers, the logistics of handling and disposal of this biological material is much less feasible and would markedly increase the workload for technical staff.

The Virtual Nitrogen Balance Experiment (VNBE)11 described here has been developed taking these considerations into account yet maintains considerable hands-on practical activity coupled with problem solving and obviates biosafety requirements. In the VNBE, the students experience both the practical aspects of data generation and collection as well as data analysis and interpretation of the results. Students are given multiple opportunities for practicing pipetting skills, calculating and carrying out dilutions, and recording data, all of which are standard important skills required of biochemistry students [2]. All analyses are based on specific colorimetric assays, and so the only major equipment needed is a visible spectrophotometer. Preparation of standard curves, use of enzymes as reagents, and statistical analysis can also be built into the objectives.

The VNBE has been developed based on our previous experience in running a similar experiment with real samples. The original experiment required students to volunteer as subjects and to consume diets estimated to be high, medium, or low in protein for 3 days, take a 3-day dietary record, and determine their dietary protein intake using computerized food composition tables, collect their urine for 24 h, and then analyze their own samples. However, students were not always willing to volunteer as subjects mainly because of the difficulty and embarrassment of collecting a 24-h urine sample so samples were not always collected for the full period. In addition many errors were inherent in the estimation of the dietary protein intake so that after completing the analyses of the urine for 24-h nitrogen excretion, nitrogen balance was rarely observed, and so this practical failed to reinforce this key concept. Subsequently the biosafety issues made a change to this format imperative.

The format for VNBE is still a project-style laboratory, and we are able to cater to large classes by running multiple streams for 3-h sessions each week. In the first week the students review the steps in designing an experiment to determine the effect of protein intake on nitrogen balance, complete their own protocol tables, and commence analysis of the urine samples for urea, uric acid, and creatinine. Analysis is completed in week two, and in week three they collate the results and also discuss and answer questions related to a research paper about nitrogen balance of patients recovering from surgery [3]. Individuals prepare a written report from their work to be submitted for formative assessment. These classes could be done in a shorter time frame, even within a single 3-h session, but one of our goals is to allow the students plenty of time to repeat an analysis if an error is made or a sample value falls beyond the range of the standard curve. The students could carry out the entire exercise individually, but we have found that a combination of individual and group work gives students the experience of working as a team. Each individual of the group is responsible for one assay and analyzes all six samples, and then groups of 3 students collate all their data for interpretation.

The VNBE together with additional discussion is thus aimed to reinforce knowledge about how various nitrogen-containing compounds in urine vary with diet and various physiological conditions. An additional component, Biochemical Detective, was developed to introduce some mystery into the project and give the students a chance to try out their problem-solving skills and apply the knowledge obtained from the VNBE. In Biochemical Detective the students are given the following scenario: a researcher has collected 24-h urine samples from three interesting subjects to study their nitrogen metabolism; unfortunately a mishap occurred and the labels have peeled off the bottles containing urine samples from the different subjects (see Fig. 1). Students are asked to deduce which label (A, B, C) matches which sample (X, Y, Z) by analyzing the samples for the nitrogen-containing compounds.

BACKGROUND THEORY

Unlike glucose and fatty acids, amino acids do not have a dedicated storage form equivalent to glycogen or fat but must be obtained daily from dietary protein to maintain the pool of amino acids. A healthy adult will be in nitrogen balance with the amount of nitrogen ingested balanced by that excreted. Dietary protein is digested, the amino acids are absorbed across the gut, released into the portal vein, and carried to the liver for energy metabolism or biosynthetic processes. Some amino acids are also redistributed to other tissues to meet similar needs. Amino acids are first deaminated, and then the carbon skeletons are metabolized. The principal mechanism for deamination is via transamination, involving the transfer of the amino group to a suitable α-keto acid acceptor. Much of the carbon flow that occurs between skeletal muscle (and several other tissues) and liver occurs by the release of alanine and glutamine into the blood by peripheral tissues and their uptake by the liver. The alanine taken into the liver is converted to pyruvate, and subsequently the nitrogen is transferred to α-ketoglutarate to form glutamate; ultimately this nitrogen is incorporated into urea. The pyruvate can be used in gluconeogenesis to produce glucose, which may be released into the blood for transport back to peripheral tissues. Glutamine may be deaminated in the kidney tubules by glutaminase and glutamate dehydrogenase to generate ammonia, which is excreted directly into the urine; this process is important in acidosis. Amino acids are also involved in the synthesis of purine and pyrimidine nucleotides, and purine degradation in humans produces uric acid, which is excreted in urine. Overproduction of purine nucleotides also causes high levels of uric acid in blood and urine. Creatine is synthesized from three amino acids, glycine, arginine, and methionine. Phosphocreatine acts as an energy store in muscle. Phosphocreatine can spontaneously lose phosphate in a cyclization reaction that generates creatinine. Because the amount of phosphocreatine is proportional to the muscle mass of the body, the formation of creatinine is characteristic of each individual and is generated at a constant rate. All of the creatinine is excreted in the urine. Good supporting descriptions of amino acid metabolism can found in most biochemistry textbooks [4, 5], and these can be supplemented with mini-reviews [6, 7]. Some of these aspects of nitrogen metabolism can be explored using the VNBE.

EXPERIMENTAL PROCEDURES

Equipment

Visible range spectrophotometer and 3-ml cuvettes; pipettors, P20, P200, P1000; vortex mixer; 37 °C waterbath; and test tubes are used. It is also useful to have bottle top dispensers for the assay reagents when the same volume needs to be dispensed repeatedly.

Simulated Urine Samples

Each simulated urine sample is prepared by dissolving the compounds in 1 liter of distilled water as described in Table I. The concentrations of the compounds in these solutions represent those that could be expected in 2 liters of urine collected over 24 h from various human subjects. Samples are diluted 1:20 by the students for the assays.

Solutions for Assays

The quantities given are sufficient for a class of about 100 students and can be scaled up or down as required. Appropriate aliquots of the solutions are supplied to each student. Analytical grade reagents should be used. All solutions are stable for at least 2 months when stored as indicated. Standard safe laboratory practice should be adhered to during handling of all chemicals and solutions.

For Urea Assay—

Urease/sodium nitroprusside (116 mM Na2EDTA, 6 mM sodium nitroprusside, 3000 units/liter urease). Dissolve 21.6 g of Na2EDTA and 0.9 g of sodium nitroprusside in 800 ml of distilled water, pH to 7.4 with NaOH, and then add 1500 units of urease (Sigma U1500) and make up to 500 ml. Store in the dark at 4 °C.

Phenol (120 mM)–Dissolve 79 g of phenol crystals in 7 liters of distilled water (take care; wear a mask and gloves). Store in dark glass bottles at 4 °C.

Hypochlorite solution–27 mM sodium hypochlorite, 0.14 M sodium hydroxide. Dissolve 31 g of sodium hypochlorite (liquid) and 39.2 g of NaOH in 7 liters of distilled water. Store in dark glass bottles at 4 °C.

Urea standard solutions–Dissolve 0.5 g of urea in 500 ml of distilled water. Dilute this 1 mg/ml stock solution to prepare four standards: 0.25, 0.5, 0.75, and 1 mg/ml.

For Uric Acid Assay—

Sodium carbonate (1.32 M). Dissolve 28 g of sodium carbonate (anhydrous) in 2 liters of distilled water. Store in plastic bottle at room temperature.

Phosphotungstate reagent–Dissolve 80 g of sodium tungstate in 600 ml of distilled water, add 64 ml of 82% H3PO4 and glass beads, and reflux gently for 2 h. Cool, dilute to 2 liters, and then add 64 g of LiSO4·H20. Store at 4 °C.

Standard uric acid stock–1 g/liter. Dissolve 0.5 g of uric acid and 0.3 g of lithium carbonate in 75 ml of distilled water. Then add 108 ml of 37% formaldehyde, 250 ml of distilled water, and 1 ml of 0.5 M H2SO4 and make up to 500 ml with distilled water. Store in plastic bottle at 4 °C. For 50 μg/ml standard, freshly dilute 20-fold with distilled water.

For Creatinine Assay—

Picric acid solution (10 g/liter). Weigh ∼30 g of picric acid (Caution: adhere to appropriate procedures for storage and handling of solid picric acid; dry crystals of picric acid may explode, so do not remove all the water when weighing) and dissolve in 3 liters of distilled water. Students then prepare their own alkaline picrate when required by mixing 30 ml of picric acid and 4 ml of 1.25 M NaOH.

Creatinine standard—(100 μg/ml). Dissolve 0.1 g of creatinine in 1 liter of 0.1 M HCl, and store in plastic bottle at 4 °C.

Protocol for Urea Assay

This method combines the use of an enzyme as a reagent with the ease of a colorimetric method. Urea is hydrolyzed to ammonia by the enzyme urease. Ammonia is then measured spectrophotometrically by the Berthelot reaction that consists of two steps involving the addition of phenol and sodium hypochlorite to generate indophenol blue, which absorbs at 546 nm [8]. Prepare a standard curve to 20 μg from the urea standards, and analyze duplicate 20-μl samples that have been diluted 1:20. This step requires very accurate pipetting and care when using phenol; safety glasses should be worn. To the standards and samples add 100 μl of urease solution; mix and incubate at 37 °C for 10 min. Then add 2.5 ml of phenol solution and 2.5 ml of hypochlorite solution; mix and incubate at 37 °C for 15 min. Read absorbance at 546 nm.

Protocol for Uric Acid Assay

Uric acid is oxidized to allantoin and CO2 by phosphotungstate in alkaline solution. Phosphotungstate is reduced in this reaction to tungsten blue, which absorbs at 710 nm [9]. Prepare a standard curve to 50 μg using the 50 μg/ml uric acid standard, analyze duplicate 1.0-ml aliquots of the diluted samples, and make up to 2.0 ml with water. To the standards and samples add 1.0 ml of sodium carbonate, 1 ml of phosphotungstate; mix and incubate at 37 °C for 15 min. Read absorbance at 710 nm.

Protocol for Creatinine Assay

This assay depends on the formation of a red chromophore, creatinine picrate, which absorbs at 520 nm, produced from the reaction of creatinine and picrate in alkaline solution [10]. Prepare a standard curve to 100 μg using the 100 μg/ml creatinine standard, and analyze duplicate 1.0-ml aliquots of the diluted samples. To the standards and samples add 2 ml of alkaline picrate; mix and incubate at room temperature for 10 min. Add 7.0 ml of water to each tube; mix and read absorbance at 520 nm.

RESULTS

VNBE—

The students were asked to find out the nitrogen balance status during the different diets and which of the nitrogen-containing compounds in urine is most affected by the protein content of these three diets. The students analyzed the three urine samples simulated to be from a subject on the different diets (high, medium, and low protein diet) to find out experimentally how the variation in dietary protein affects the level of nitrogen-containing compounds excreted in a 24-h urine sample.

The students prepared standard curves to convert the absorbance of the corresponding assay samples into the amount of the nitrogen-containing compound in the samples. Then the amount present in the 2-liter, 24-h urine sample was then calculated, taking into account the volume assayed and dilution factor.

x μg/volume of aliquot assayed (ml) = x μg/ml of diluted sample

(x μg/ml of diluted sample) × (dilution factor [20]) × (volume of urine in 24 h (2 liters))

From this the total amount of nitrogen (g/24 h) was determined, using the proportion of nitrogen present in the nitrogen-containing compound.

(x μg/ml × dilution factor × volume of urine in 24 h) × g nitrogen (in 1 mole)/Mr = g nitrogen/24 h

The data obtained by students carrying out the VNBE are shown in Table II. Statistically significant differences between diets have been indicated. The coefficients of variation (S. D./mean × 100 (%)) for the repeated measurements were as follows: for urea, 8, 28, 33; for uric acid, 10, 12, 14; and for creatinine, 20, 19, 17 for the high, medium and low protein samples, respectively. The students interpreted their data and could conclude the following: normal healthy subjects on high, medium, and low protein diets remain in nitrogen balance; the major component that changes is the urea; uric acid, which arises primarily from nucleotide breakdown from DNA, tends to be higher on the high protein diet because meat normally consumed on these diets contains more nuclear material; and creatinine does not change because it is proportional to the muscle mass of the subject.

Biochemical Detective—

The results obtained by the students for the “mystery samples,” X, Y, and Z, for the Biochemical Detective section of the exercise are shown in Table III. From these results the students deduced which sample comes from which subject as described on the labels (see Fig. 2). The reasoning used is as follows. Because healthy adult people, i.e. subjects A and C, are in nitrogen balance, and subject A was consuming 35 g of protein, sample Y belongs to A. Solely from nitrogen excreted, either sample X or Z could match the healthy football player. Additional features therefore need to be considered to distinguish between these two. Although subjects B and C are about the same body weight, the muscle mass of the football player, C, would be expected to be greater than that of the obese subject, B, where adipose tissue would be contributing substantially to the subject's body weight. Therefore sample X was from the football player because of the higher creatinine level. This suggests that sample Z was from the subject undergoing the fast, B, who is in negative nitrogen balance. The higher level of nitrogen excreted as ammonia in sample Z is consistent with fasting for about 5 days when acidosis occurs because of the synthesis of ketones. In addition, although subject B is much heavier than subject A, their creatinine excretions are similar because their muscle masses must be similar.

DISCUSSION

This VNBE has proved to be a successful laboratory experiment that avoids the biosafety issues of urine collection, handling, and disposal and could easily be used in many biochemistry courses for multiple streams of large classes. It is of interest that other exercises using simulated urine have been described [11, 12]; these also avoid any possible health risks and overcome ethical issues of using animals for teaching. In one exercise [12], guarurine, a simulated urine containing various levels of glucose and ketones representative of different patients, has been proposed to teach urine analysis of diabetics. In the other [11], samples are simulated to mimic effects of acid-base balance and students determine titratable acidity. We envisage that these exercises together with ours could form the basis of a comprehensive suite of simulated exercises that covers many aspects of metabolism.

Another advantage of this VNBE is that it obviates the inherent errors of estimation of dietary protein intake from dietary records and in the collection of 24-h urine samples. We have found that in class experiments these errors were extremely difficult to minimize and control for. The VNBE overcomes the frustrations that students had previously with determination of nitrogen balance with sufficient accuracy to draw meaningful conclusions. The precision with which students could obtain the same value for a single sample was not very high, even for the VNBE as shown by a median coefficient of variation of 17% (range 8–33%), and is probably a reflection of the inexperience of the students with these new techniques.

The VNBE combines the advantages of problem-based learning with the need for biochemistry students to become proficient in laboratory skills. Throughout the experiment students have many opportunities to practice pipetting, prepare standard curves, follow assay protocols, use spectrophotometers, and record data. It is our observation that the students take great pride in producing reproducible standard curves and duplicates, willingly repeat assays if necessary, and actively participate in discussion of the results. In addition, the collation of all the individual results generated by the students could be used to introduce the concepts of error and statistical measures of precision (e.g. calculations of standard deviation, coefficient of variation) and other statistical methods such as Student's t test to determine whether two values are significantly different. Overall the students derive new knowledge about the metabolism of amino acids from data that they have generated and analyzed themselves. Other scenarios for Biochemical Detective could be easily devised using data from the research literature, for example, for a body builder [13], a patient recovering from surgery [3], or a lactating woman [14], depending on the interests of the student cohort.

Figure FIGURE 1..

Labels from urine sample bottles for three subjects.

Figure FIGURE 2..

Solution to the Biochemical Detective problem.Arrows indicate which samples were determined to belong to which subjects.

Table Table I. Composition of simulated urine samples
 High proteinMedium proteinLow proteinXYZ
  • a

    a Food color (102) added to give the appearance of straw-colored urine.

Make up to 1 liter with distilled water.
Urea (g)19.59.03.329.03.07.0
Uric acid (g)0.750.60.450.70.40.4
Creatinine (g)0.910.910.911.00.80.8
Lithium carbonate (g)0.60.60.60.60.60.6
Yellow food coloringa (μl)200200200200200200
Table Table II. Collated class results of virtual experiment on the effect of diet on nitrogenous compounds in urine
Level of protein in dietHighMediumLow
  • a

    a Data provided to students.

  • b

    b Assumed 1.5 g nitrogen/24 h lost via sweat, feces, shed skin, etc.

Values for urea, uric acid and creatinine are given as mean ± S.D. from the same samples analyzed by n students, and means not sharing a common superscript are significantly different at p < 0.01 by ANOVA. Nitrogen balance is determined by subtracting the total nitrogen out (bold) from total dietary intake of nitrogen (bold).
Amount of protein in diet140 g70 g35 g
Total dietary intake of nitrogen (g/day; 16% of dietary protein)22.411.25.6
Urea nitrogen (g/24 h) n = 5218.1 ± 1.397.68 ± 2.15§2.98 ± 0.99
Uric acid nitrogen (g/ 24 h) n = 410.47 ± 0.050.39 ± 0.05§0.21 ± 0.03
Creatinine nitrogen (g/24 h) n = 380.65 ± 0.130.66 ± 0.130.65 ± 0.11
Ammonia nitrogena (g/24 h)0.70.50.16
Other losses of nitrogenb (g/24 h)1.51.51.5
Total nitrogen out (g/24 h)21.410.75.5
Table Table III. Collated results of Biochemical Detective
 UreaUric acidCreatinineAmmoniaaOther lossTotalProtein equivalentb
  • a

    a Values supplied (the students received the information that “the ammonia levels had been measured on fresh samples and the results recorded on the bottle.”

  • b

    b Assuming protein is 16% nitrogen.

Values for urea, uric acid, and creatinine are given as mean ± S.D. and are from the samples analyzed by n students, and means not sharing a common superscript are significantly different at p < 0.01 by ANOVA.
 n = 52n = 41n = 38    
   g nitrogen/24 h   g/day
X8.26 ± 1.350.42 ± 0.040.72 ± 0.151.01.511.974.4
Y2.66 ± 0.58§0.19 ± 0.03§0.59 ± 0.12§0.51.55.4434
Z6.5 ± 1.250.17 ± 0.040.60 ± 0.163.01.511.873.6

Footnotes

  1. 1

    The abbreviation used is: VNBE, Virtual Nitrogen Balance Experiment.

Ancillary