Urinary protein and albumin excretion corrected by creatinine and specific gravity

Clinica Chimica Acta 294 (2000) 139–155 www.elsevier.com / locate / clinchim

Urinary protein and albumin excretion corrected by creatinine and specific gravity a b c, David J. Newman , Michael J. Pugia , John A. Lott *, Jane F. Wallace b , Andrew M. Hiar b a

SW Thames Institute for Renal Research, St. Helier Hospital, Wrythe Lane, Carshalton, Surrey SM5 1 AA, UK b Diagnostics Business Group, Bayer Corporation, 1884 Miles Avenue, Elkhart, IN 46514, USA c Department of Pathology, The Ohio State University Medical Center, Starling Loving M-029, Columbus, OH 43210, USA Received 17 September 1999; received in revised form 3 December 1999; accepted 17 December 1999

Abstract Timed urine collections are difficult to use in clinical practice owing to inaccurate collections making calculations of the 24-h albumin or protein excretion questionable. One of our goals was to assess the ‘correction’ of urinary albumin and (or) protein excretion by dividing these by either the creatinine concentration or the term, (specific gravity 2 1) 3 100 1 . The 24-h creatinine excretion can be estimated based on the patients’ gender, age and weight. We studied the influence of physiological extremes of hydration and exercise, and protein and creatinine excretion in patients with or suspected kidney disorders. Specimens were collected from healthy volunteers every 4 h during one 24-h period. We assayed the collections individually to give us an assessment of the variability of the analytes with time, and then reassayed them after combining them to give a 24-h urine. For all volunteers, the mean intra-individual CVs based on the 4-h collections expressed in mg / 24 h were 80.0% for albumin and 96.5% for total protein (P . 0.2). The CVs were reduced by dividing the albumin or protein concentration by the creatinine concentration or by the term, (SG-1) 3 100. This gave a CV for mg albumin / g creatinine of 52% (P , 0.1 vs. albumin mg / g creatinine); mg protein / g creatinine of 39% (P , 0.05 vs. mg protein / g creatinine);

Abbreviations: AER, albumin excretion; PER, protein excretion; ACR, albumin creatinine ratio; PCR, protein / creatinine ratio; ASG, albumin /(SG-1)3100 ratio; PSG, protein /(SG-1)3100 ratio; TP, true positive; FP, false positive; TN, true negative; FN, false negative; Alb, albumin; Cre, creatinine; SGU, (1-SG)3100 *Corresponding author. Tel.: 11-614-293-5383; fax: 11-614-293-5984. E-mail address: [email protected] (J.A. Lott) 1 Note that the term, (SG-1)3100 increases with the increasing concentration of all dissolved solids. Because (1-SG)3100 is dimensionless, we called (SG-1)3100 ‘SG units’, i.e. SGU. 0009-8981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00181-9

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mg albumin / [(SG-1) 3 100] of 49% (P , 0.1 vs. albumin) / [(SG-1) 3 100]; and mg protein / [(SG1) 3 100] of 37% (P , 0.05 vs. mg protein) / [(SG-1) 3 100]. For the 68 subjects in the study, the strongest correlation was between the creatinine concentrations and the 24-h urine volume: r 5 0.786, P , 0.001. The correlation of (SG-1) 3 100 vs. the 24-h urine volume was: r 5 0.606, P , 0.001; for (SG-1) 3 100 and the creatinine concentration, the correlation was: r 5 0.666, P , 0.001. Compared to the volunteers, the albumin and protein excretion in mg / 24 h were more variable in the patients. The same was true if the albumin or protein concentrations were divided by the creatinine concentration or by (SG-1) 3 100. Protein and albumin concentrations were lower in dilute urines. Dividing the albumin or protein concentrations by the creatinine concentration reduced the number of false negative protein and albumin results. Dividing the albumin or protein values in mg / 24 h by (SG-1) 3 100 eliminated fewer false negatives. Albumin concentrations increased significantly after vigorous exercise. The increase was almost eliminated when the albumin result was divided by the creatinine concentration suggesting that a decreased urine flow and not increased glomerular permeability causes an increase of post-exercise albuminuria. The same was true for proteinuria. A dipstick test plus an optical strip reader that can measure urine protein, albumin, and creatinine and calculate the appropriate ratios provides a better screening test for albuminuria or proteinuria than one measuring only albumin or protein.  2000 Elsevier Science B.V. All rights reserved. Keywords: Albuminuria; Effects of exercise; Microalbuminuria; Proteinuria; Renal failure; Specific gravity; Test variation

1. Introduction Urine dipstick testing is widely used for the detection of early proteinuria and possible early renal damage [1]. There are a variety of analytical methods available for the measurement of both urine total protein and albumin concentrations [2–4]. They differ in sensitivity and specificity for the proteins in urine, and their relative analytical and clinical performances have been evaluated in this context. The analytical measurement of protein may be simple, but the estimation of the amount excreted in 24 h is fraught with error. The volume of urine excreted can be highly variable depending mainly on the individual’s fluid intake and physical activity. In a dilute urine, the total protein excretion may be underestimated. If the urine is concentrated, as frequently occurs after strenuous physical activity, an increased protein concentration could be misinterpreted. To avoid this problem, accurately timed urine specimens have been proposed, expressing protein excretion in units of mg / min. The difficulty is in collecting an accurate 24-h specimen [5–7]. To reduce the uncertainty of the timing, a collection is made of the early morning first voiding [1]. But a means of correcting for urine concentration and (or) volume is needed when the collection accuracy is in doubt. Two techniques used to compensate for variation in the excretion volumes are to divide the albumin or protein concentrations by the creatinine concentration or by the term, (specific gravity 2 1) 3 100 [8].

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Urinary creatinine excretion in normal persons depends on gender, age, and muscle mass [9,10]. The 24-h creatinine excretion in a patient with stable glomerular filtration is fairly constant [11], and is the basis of the Cockroft– Gault calculation for creatinine excretion as is given here [12]. The ratios, milligram of albumin or milligram of protein per gram of creatinine, are readily determined. If we calculate the creatinine excretion per 24 h, then the urinary albumin and (or) protein loss are easily calculated. An estimate of the 24-h albumin (or protein) excretion is given by: (g creatinine / 24 h) 3 (mg albumin / g creatinine) 5 mg albumin / 24 h. The first term is measured or calculated from the Cockroft–Gault equation, and the second term is obtained from the assay of albumin (or protein) and creatinine. The error in this approach is much smaller than the typical errors made in ‘24-h urine’ collections. Various pathologies influence the urinary creatinine excretion. It is often decreased in advanced renal disease, acromegaly, strenuous exercise, hyperthyroidism and muscle damage potentially causing an underestimation of protein excretion. Creatinine excretion is commonly increased in diabetes mellitus and hypothyroidism causing overestimation of protein excretion [10]. A few studies compared creatinine and specific gravity as correction factors when an incomplete collection occurs. The term ‘correcting for SG’ indicates dividing the albumin or protein concentration by (SG-1)3100 [10]. The urinary excretion of creatinine has been used for many years to get an estimate of the 24-h excretion volume and to ‘correct’ the quantitative measurement of other analytes such as albumin, proteins, cortisol, and catecholamines to a 24-h basis [13,14]. Until recently, no point of care or dipstick tests were available to make the correction [15,16]. A combined measurement dipstick is now available on the CLINITEK  50 instrument (Bayer Corp., Tarrytown, NY) that uses novel analytical techniques for both albumin and creatinine. These methods are more accurate when read by a reflectometer rather than visually [15–17]. We used these dipsticks to assess the utility of dividing the results by creatinine or (SG-1)3100 for adjusting results on random urines. One of our goals was to measure intra- and inter-individual variation of urinary albumin and protein concentrations in healthy individuals and in patients with impaired renal function. Comparisons were made between excretion in mg / 24 h and the ratios of albumin or protein to creatinine or to (SG-1)3100. The correlation of urinary volumes to creatinine or (SG-1)3100 was calculated from the 24-h collection data of the healthy volunteers and patients. We also tested other healthy individuals following strenuous exercise with physical contact. The latter commonly leads to dehydration and overestimation of albumin or protein concentrations. Finally we determined the utility of a new

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urinalysis strip for albumin, protein, and creatinine by comparison to quantitative methods.

2. Materials and methods

2.1. Healthy volunteers A total of 118 specimens were collected during one 24-h period at 4 h intervals from 20 healthy volunteers. Some of the volunteers were awakened if needed to provide a urine specimen during their sleeping time. One subject provided only four specimens. We also recorded the gender, age and weight of the volunteers; these are required for the Cockcroft–Gault equation [12]. We performed quantitative assays for albumin, protein, creatinine, and measured the volumes for all individual collections in a 24-h period; these were then combined to simulate a 24-h urine collection. SG was determined by refractometry. An additional 19 specimens were collected from 16 of the same volunteers after 12 h of fluid deprivation. Three volunteers gave us specimens after 12 and 14 h of fluid deprivation. The volunteers then drank 1 l of water, and the first voiding was collected 1 h later from 12 volunteers and assayed as above.

2.2. Hospitalized patients A total of 68, 24-h urine specimens were collected from patients with confirmed or likely kidney disorders. Among these, 34 had chronic renal failure with and without nephrotic syndrome, previous kidney transplant, or diabetic nephropathy. Six had hypertension without diabetes or known kidney disorder but spilled an abnormal amount of protein. Eighteen had diabetes mellitus with renal insufficiency, and 10 had cancer and a serum creatinine .20 mg / l that suggested renal insufficiency.

2.3. Football players The effect of exercise on the urinary concentrations of creatinine and albumin was determined on specimens from 61 members of the varsity football team at the University of St. Francis (Joliet, IL), 36 participated in a 2-h football game (players), and 25 sat on the bench during the entire game (non-players). Spot urine specimens were collected just prior to the game and within 1 h after the game from all 61 subjects. The specimens were analyzed immediately for albumin and creatinine both quantitatively and with the new dipsticks on a Bayer CLINITEK 50 analyzer.

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2.4. Quantitative laboratory methods For albumin, we used the Dade–Behring immunonephelometric method in their Analyzer II instrument (Dade Behring, Miami, FL). For creatinine, we used a kinetic Jaffe method in the Cobas Mira analyzer (Roche Diagnostic Systems, Sommerville, NJ) with the reagents and procedure from Roche. For protein determinations, the Sigma (St. Louis, MO) Microprotein pyrogallol red method was performed in the Mira analyzer according to the manufacturer’s instructions. Specific gravity was determined using a TS meter (Cambridge Instruments Inc., Buffalo, NY). Corrections for specific gravity were calculated by dividing the mg / l albumin or protein by the term, (SG-1)3100. Specimens were refrigerated at 48C and tested the next day or frozen on the day of collection and stored for up to 14 days, thawed overnight at 48C, and assayed. The above tests were performed in duplicate on each specimen. Pre- and post-game specimens from all 61 football team members were assayed only once for the above tests.

2.5. Point-of-care methods The Bayer DCA-2000 1 Analyzer was used for the quantitative determination of albumin and creatinine in pre- and post-exercise urine specimens. This point-of-care analyzer permitted on-site testing with results available in 5 min. Duplicate results were also obtained for all specimens using a new urinalysis dipstick from Bayer that has test pads for albumin, protein and creatinine. The strips were read in a CLINITEK 50 reflectometer [16]. This instrument provides semiquantitative results for albumin and gives readings of 10, 30, 80, 150 and 300 mg / l; the creatinine pads give values of 100, 500, 1000, 2000 and 3000 mg / l; and the protein pads give readings of 0 or negative, 150 or trace, 300, 1000 and 3000 mg / l. The Clinitek 50 dipstick reader gives only one of the values stated above for each test and no in-between results.

2.6. Statistical analyses Nonparametric comparisons were performed using the Mann–Whitney U-test curve-fitting algorithm (Microsoft Excel) to obtain the equations and coefficient of determination (R 2 ) for the line of best fit for urine creatinine vs. urine volume and (SG-1)3100 vs. urine volume. The Cockroft–Gault equations for estimating the 24-h creatinine excretion are [16]: • For men, creatinine5[(1402age in years)(weight in kg)] / 5000. • For women, creatinine5[(1402age in years)(weight in kg)]30.85 / 5000. For example, application of the equation to a 60-year-old man weighing 70 kg gives a 24-h creatinine excretion of [(140260)3(70)] / 500051.12 g.

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3. Results

3.1. Intra-individual variation in healthy adults Fig. 1 shows the intra-individual variations in albumin, protein, creatinine and SG in the 4-h collections as determined in 20 healthy volunteers during a 24-h period. The concentrations were calculated based on quantitative results and expressed as mg / 24 h, mg / g creatinine, or mg / [(SG-1)3100]. Statistical comparisons are in the legend for Fig. 1.

3.2. Inter-individual variation of all subjects Mean excretions including ranges and inter-individual variations for 24-h collections in our five groups are shown in Table 1. The creatinine excretion was

Fig. 1. Mean intra-individual variation in urine parameters in 20 healthy volunteers during a 24-h period. Albumin excretion vs. protein excretion, P,0.2; albumin excretion vs. albumin / creatinine ratio, P,0.1; protein excretion vs. protein / creatinine ratio, P,0.1; albumin excretion vs. albumin / SGU ratio, P,0.05; protein excretion vs. protein / SGU ratio, P,0.05.

Table 1 Inter-individual variation in urinary parameters for creatinine, SG, volume, albumin, and protein

Creatinine (mg/l) Creatinine (mg/24 h) Specific gravity Urine volume (l) Albumin AERh (mg/24 h) Albumin ACRh (mg/g) Albumin ASG h (mg/SGU) Protein PERh (mg/24 h) Protein PCRh (mg/g) Protein PSG h (mg/SGU) a

Avg.c (Range) %CV c Avg. (Range) %CV Avg. (Range) %CV Avg. (Range) %CV Avg. (Range) %CV Avg. (Range) %CV Avg. (Range) %CV Avg. (Range) %CV Avg. (Range) %CV Avg. (Range) %CV

Healthy (n520)

Kidney damage (n534)

Hypertension (n56)

Diabetes (n518)

Cancer a (n510)

1083 (412–2562) 55 1638 (1114–3697) 26 1.015 (1.007–1.031) 64 1.90 (0.44–3.61) 46 6.1 (2.2–10.1) 34 3.9 (1.3–8.0) 42 2.6 (1.0–5.2) 45 79 (38–179) 38 50 (26–108) 40 33 (19–63) 32

545 d (163–1496) 48 1162 e (235–2667) 43 1.010 (1.003–1.021) 41 2.33 (0.45–4.17) 38 783 d (4.2–4289) 142 660 d (3.4–2875) 120 376 d (2.2–1895) 123 1743 g (50–8169) 104 1895 d (55–16527) 150 889 d (29–4107) 102

1008 (311–1926) 51 1248 (230–1908) 47 1.015 (1.008–1.023) 54 1.33 (0.62–2.45) 51 125 (3.9–525) 153 407 (3.3–2283) 215 163 (2.7–888) 208 358 e (83–779) 83 739 (72–3388) 170 325 (53–1317) 146

600 e (165–1927) 66 1636 (45–11735) 155 1.015 (1.005–1.032) 65 2.36 (0.90–4.26) 55 697 f (6.3–4780) 185 1033 f (1.3–4736) 155 352 e (0.8–2007) 172 1357 f (57–7961) 153 1850 f (26–8025) 141 638 f (16–3400) 154

508 d (189–1357) 65 1112 d (783–1686) 25 1.012 (1.006–1.023) 57 2.88 (0.88–4.67) 46 410 f (9.1–2163) 148 379 f (5.4–1965) 144 187 (1.8–1046) 159 1282 d (198–3119) 70 1302 d (117–3116) 76 492 d (40–1509) 84

All b (n588) 67 90 59 48 195 183 178 143 173 144

Patients with cancer and serum creatinine concentrations of $20 mg / l. b The percentage CV of 24-h specimens from all 88 subjects, i.e. the healthy volunteers and the patients. c The average and percentage CV observed with 24-h specimens for a group of healthy volunteers and for different groups of hospitalized patients with known or likely kidney disorders. d P,0.05 vs. Healthy. e P,0.1 vs. Healthy. f P,0.2 vs. Healthy. g P,0.01 vs. Healthy. h Abbreviations: AER, albumin excretion; PER, protein excretion; ACR, albumin / creatinine ratio; PCR, protein / creatinine ratio; ASG, albumin /(SG-1)3100 ratio; PSG, protein /(SG-1)3100 ratio.

D. J. Newman et al. / Clinica Chimica Acta 294 (2000) 139 – 155

Urinary parameters (units)

145

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significantly lower in patients with kidney diseases, diabetes, and cancer. Creatinine concentrations varied inversely with the urine volume, as expected (Fig. 2a). The fit of the points to the curvilinear regression line was good, and

Fig. 2. (a) Relationship of 24-h urine volume and urine creatinine concentration in healthy volunteers and patients. R 2 50.618, r50.786, P,0.001, n588. (b) Relationship of 24-h urine volume with urine (SG-1)3100 in healthy volunteers and patients. R 2 50.367, r50.606, P, 0.001, n588.

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147

the number of outliers can be judged from Fig. 2a. The term, (SG-1)3100 was a less efficient predictor of urine volume (Fig. 2b). In order to test the relationship between the urine volume and creatinine, specimens with creatinine concentrations of ,250 mg / l were segregated, and the albumin, protein, specific gravity and volume were determined. Creatinine concentrations of ,250 mg / l were observed in only 10 patients with kidney disorders (Table 2). Five of the 10 had albumins of ,30 mg / l, and four of these had normal albumins when the albumin was divided by the creatinine. Nine of the 10 negatives had a normal protein, i.e. ,150 mg / l, and the remaining patient had a normal protein when divided by creatinine. Creatinine concentrations of $2500 mg / l were observed in two of the 20 volunteers; none of the 68 patients had such high values. Summaries of the data from the volunteers (‘Healthy’) and all the patients are given in Table 1.

3.3. Agreement of dipstick results with quantitative assays The agreement of the semiquantitative dipstick results with the quantitative laboratory results for albumin, creatinine, and protein was tested with specimens from the volunteers and patients. All assays for albumin, protein and creatinine were performed in duplicate. The albumin and protein data are shown in Tables 3 and 4. All but one of the dipstick tests on the healthy volunteers was classified as normal for albumin. The data for the 68 patients from Tables 3 and 4 along with sensitivity and specificity calculations are summarized in Table 5. For both albumin and protein concentrations expressed in the three ways, the sensitivities were not statistically different for any of the comparisons (P.0.05). Two of the specificities were statistically different (Table 5).

3.4. Effect of reduced fluid intake on creatinine and SG in healthy volunteers The creatinine and SG for 16 healthy volunteers were determined after 12 h of fluid depravation that increased the creatinine concentrations by an average of 1587 mg / l. With the quantitative method, creatinines in the subjects were all .750 mg / l with 47%.1500 mg / l, and 25%.2000 mg / l. One subject had a urine creatinine concentration of 3980 mg / l, our highest value. By dipstick, 63% were $2000 mg / l. SG was also increased to $1.020 in 16 out of 19 specimens. All specimens collected within 1 h following hydration with 1 l of water had creatinine values of ,920 mg / l with an average of 493 mg / l. By the dipstick, all were #500 mg / l. SG was not significantly reduced (P.0.05) and was only ,1.010 in two out of 12 cases. The 24-h urines had an average quantitative

148

Condition

Urinary albumin concentration

Urinary protein concentration

Creatinine

Albumin

AER

ACR

ASG

Protein

PER

PCR

PSG

(mg/l)

(mg/l)

(mg/24 h)

(mg/g)

(mg/SGU)

(mg/l)

(mg/24 h)

(mg/g)

(mg/SGU)

a

567

a

Kidney damage

163

Neg.

35

148

34

Neg.

572

2436

Kidney damage

164

Neg.a

54

79

43

Neg.a

666

973

532

Diabetes

165

Neg.a

Neg.a

Neg.a

Neg.a

Neg.a

Neg.a

Neg.a

Neg.a

Kidney damage

175

Neg.a

46

86

Neg.a

Neg.a

614

1146

Neg.a

a

Cancer

189

69

291

365

98

Neg.

1542

1931

520

Cancer

207

32

148

153

45

Neg.a

933

964

Neg.a

a

Diabetes

213

600

1052

2817

400

Neg.

2048

5484

779

Kidney damage

232

344

1166

1484

687

Neg.a

1906

2425

1123

Kidney damage

249

218

434

877

218

411

8169

16527

4107

Cancer

249

Neg.a

60

71

Neg.a

Neg.a

2661

3116

432

a

Reference (‘normal’) ranges: ‘Neg.’ (negative) used here are defined here as: ,30 mg of albumin / l or ,30 mg albumin / g creatinine or ,300 mg of protein / l, or ,300 mg protein / g creatinine. The correction for (SG-1)3100 is expressed here as ‘mg / SGU’; i.e. the albumin or protein values were divided by the term, (SG-1)3100. Abbreviations: AER, albumin excretion; PER, protein excretion; ACR, albumin / creatinine ratio; PCR, protein / creatinine ratio; ASG, albumin / [(SG1)3100] ratio; PSG, protein / [(SG-1)3100] ratio; Neg., negative.

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Table 2 Albumin and protein concentrations for 10 patients with ,250 mg creatinine / l in a 24-h collection

Condition

Number

Albumin

Units Healthy

Patients a

a

No. quant.

No. strip

No. quant.

No. strip

No. quant.

No. strip

results

results

results

results

results

results

,30

,30

30–149

$150

30–149

,30

$150

,30

30–149

b

30–149

$150

$150

20

mg/l

19

19

0

0

1

0

1

0

0

0

0

20

mg/24 h

20

20

0

0

0

0

0

0

0

0

0

0 0

20

mg/g c

20

19

1

0

0

0

0

0

0

0

0

0

68

mg/l

25

18

5

2

15

2

8

5

28

0

5

23

68

mg/24 h

19

15

3

1

23

4

15

4

26

1

4

21

68

mg/g c

20

18

2

0

20

3

11

6

28

0

4

24

Patients with various disorders as given in Table 1. For example, of the 20 subjects in the Healthy group, 19 had a quantitative albumin of ,30 mg / l, and negative dipstick values. One Healthy subject had a quantitative and dipstick albumin between 30 and 149 mg / l. All tests were performed in duplicate and the results averaged. Duplicate dipsticks agreed in all cases. c Strip results in mg / g are based on ratio of the concentrations of albumin to that of creatinine. b

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Table 3 Albumin concentrations in specimens from volunteers and patients. Albumin results by quantitative laboratory methods and dipsticks a

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150

Table 4 Protein concentrations in specimens from volunteers and patients. Protein results by quantitative laboratory method and dipsticks a Condition

Number

Protein

No. quant. results

No. strip results

Units

,300

,300

$300

No. quant. results

No. strip results

$300

,300

$300

Healthy

20 20 20

mg/l mg/24 mg/g b

20 20 20

20 20 20

0 0 0

0 0 0

0 0 0

0 0 0

Patients with conditions

68 68 68

mg/l mg/24 h mg/g b

29 21 23

22 17 21

7 4 2

39 47 45

0 2 3

39 45 42

a

Number of dipstick results for 20 healthy subjects and 68 patients with various disorders. All tests were performed in duplicate and the results averaged. Duplicate dipsticks results agreed in all cases. b Strip results in milligrams per gram are based on ratio of the concentrations of protein to creatinine.

creatinine of 716 mg / l, with all values between 250 and 2500 mg / l; the dipsticks gave 500–2000 mg / l. The 24-h concentration agreed with the average of all the individual specimens as expected. The individual specimens were portions of the combined 24-h specimens. The average 24-h creatinine excretion agreed with the Cockcroft–Gault value, 615% for all specimens suggesting that these were accurately collected 24-h specimens. The albumin and protein results were not as affected by the specimen volume when the ratio to creatinine was used. Individual albumin results ranged from 0.2 to 83 mg / l. All albumin values above the reference range of .30 mg / l returned to normal after correction by creatinine. The albumin / creatinine ratios were 1.0–28.5 mg / g for all specimens. We consider those with ratios #20 mg / g Table 5 Albumin and protein concentrations in specimens from patients. Sensitivity and specificity for albumin and protein results and ratios in 68 patients a Test

Units

No. TP

No. FP

No. TN

No. FN

Albumin

mg/l mg/24 h mg/g creatinine

41 44 45

7 4 2

18 15 18

2 5 3

95.3 89.8 93.8

72.0 78.5 90.0 b

Protein

mg/l mg/24 h mg/g creatinine

39 45 42

7 4 2

22 17 21

0 2 3

100.0 95.7 93.3

75.9 81.0 91.3 c

a

% Sensitivity

TP, true positive; FP, false positive; TN, true negative; FN, false negative. For the specificity data, P,0.05 for albumin in mg / l vs. mg albumin / g creatinine. c P,0.05 for protein in mg / l vs. mg protein / g creatinine. b

% Specificity

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as borderline normal. The expected albumin excretion of 2.0–10.4 mg / 24 h matched the observed 24-h albumin excretions of 2.2–10.1 mg / 24 h. Individual protein results ranged from 0 to 364 mg / l. The average protein / creatinine ratio was similar for all specimens, i.e. 46.2–51.7 mg / g. The expected protein excretion was 40–133 mg / 24 h based on the Cockcroft–Gault calculated creatinine concentration. This agreed reasonably well with the observed 24-h protein excretion of 30–176 mg / 24 h.

3.5. The effect of exercise on albumin and creatinine excretion in football players The mean creatinine concentrations of urine specimens from football players were greatly increased by exercise from pre-game mean (S.D.) values of 1590 mg / l (680) to 3400 mg / l (870) post-game, P,0.001. For the non-players, the

Fig. 3. Albumin concentrations, creatinine concentrations and ratios of the albumin / creatinine concentrations by dipstick for 25 non-players and 36 players before and after a football game. The albumin / creatinine ratio was not significantly different (P,0.2) between the pre- or post-game values for the players or the non-players.

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mean creatinine was 1780 mg / l (600) pre-game and 1900 mg / l (1020), post-game (P.0.5). In the players, the albumin increased from 18.2 to 295.0 mg / l giving an average gain of 110.6 mg / l from pre-game to post-game (P,0.001). The non-players showed a change in the albumin ranging from 23.5 to 66.3 mg / l (average gain of 12.8 mg / l, P,0.001). The albumin / creatinine ratio reduced the effect of exercise. The players had a mean of 6.7 mg / g (12.2) before and a mean of 37.9 mg / g (27.2) post-game or a change ranging from 215.5 to 109.0 mg / g (average gain of 31.2 mg / g, P,0.005). Non-players had a mean of 4.1 mg / g (1.0) before and a mean of 14.4 mg / g (18.6) post-game or a change ranging from 21.0 to 83.0 mg / g (average gain of 8.8 mg / g, P.0.2). The dipsticks also showed increased creatinine and albumin concentrations post exercise in the players but not in the non-players (Fig. 3). All post-game albumin results of players were $30 mg / l, and most had .3000 mg / l creatinine concentrations. The albumin / creatinine ratio was not significantly different (P.0.2) between the pre- or post-game values for either players or non-players.

4. Discussion In clinical practice, it is difficult to verify that a timed collection of urine is complete [17,18]. The inter-individual creatinine excretion is variable and is affected by age, gender and muscle mass. The many causes of renal insufficiency and failure are also factors. In the patients, using the creatinine excretion gives an estimate of whether the urine is representative of a complete 24-h urine collection [9,17,18]. Intra-individual creatinine excretions are reasonably steady at a constant hydration. The uncertainty in estimating the completeness of a urine collection is about 615% [12]. Nevertheless, this is still superior to an albumin or protein assay on a random specimen. Most nephrologists insist that creatinine be determined on all random urines, and the validity of this approach is well documented [19]. In our study of a group of healthy individuals, the 24-h creatinine excretion showed greater variability than the urine volume that in turn showed greater variability than the specific gravity (Fig. 1). The creatinine excretion was also significantly different between the different patients groups and showed a wider variation than did the urine volume (Table 1). Calculating the ratios of analyte to creatinine is an imperfect tool, nevertheless, we were able to demonstrate the completeness of our collections in the volunteers by demonstrating agreement between the predicted and measured creatinine excretions. Urine specific gravity was significantly less variable than either creatinine excretion or urine volume (Table 1) Plots of creatinine vs. the urine volume

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showed less scatter (Fig. 2a) than did SG vs. the urine volume (Fig. 2b). Urine albumin excretion reflects primarily glomerular permeability, whereas total protein excretion reflects a combination of permeability, tubular leakage, tubular secretion, and normal protein in the urine shed by the kidneys, e.g. the Tamm–Horsfall protein. For the volunteers, the CV of the total protein excretion (96.5%) was not more variable than that of albumin (80.0%) (P.0.2). Howey reported a similar intra-individual variation of 103% for albumin in random urines [18]. The variability of protein and albumin excretion in different patient populations is shown in Table 2. The table shows that albumin is more variable than total protein in the patients but not in the volunteers. The manner in which protein or albumin excretion was expressed, i.e. mg / 24 h, mg / g, etc. made no consistent difference within patient groups. Exercise is known to increase the urine concentrations of albumin, protein, and creatinine. Exercise causes water loss, and the individuals typically produce a more concentrated urine that is evident from the creatinine concentration [20–24]. We observed this phenomenon in the football players that did vs. those that did not play. Our quantitative analyses confirmed previous reports that there is a degree of exercise-induced proteinuria. The proteinuria would be grossly overestimated if corrections for the decreased urinary volume were not made. The semi-quantitative dipstick test confirmed the influence of exercise. The ratio of the protein or albumin to the creatinine concentration eliminated false positive albumin and protein values owing to dehydration. In conclusion, we found for the 88 subjects in the study, the strongest correlation was between the creatinine concentrations and the 24-h urine volume: r50.786, P,0.001. The correlation of (SG-1)3100 vs. the 24-h urine volume was: r50.606, P,0.001, and the correlation for (SG-1)3100 vs. the creatinine concentration was: r50.666, P,0.001. Intra- and inter-individual variation in protein and albumin excretion were reduced by dividing by the creatinine concentration, or by (SG-1)3100. Dividing by creatinine also corrected for exercise-induced albuminuria. The dipstick results had a low incidence of false negatives. Dipsticks with protein, albumin and creatinine pads gave good agreement with the quantitative methods. The ratio of albumin or protein to creatinine improved the specificity as compared to the value in milligrams per liter. The sensitivity increased slightly, but the change was not significant.

Acknowledgements We thank Dr Donald R. Parker of Bayer Corporation, Elkhart, Indiana, for his enthusiastic support during the study.

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References [1] Murakami M. Screening for proteinuria and hematuria in school children: methods and results. Acta Paediatr Jpn 1990;32:682–9. [2] Parsons M, Newman DJ, Pugia MJ, Newall RG, Price CP. Performance of a reagent strip device for quantitation of the urine albumin: creatinine ratio in a point-of-care setting. Clin Nephrol 1999;51:228–32. [3] Parsons MP, Newman DJ, Newall RG, Price CP. Validation of a point-of-care assay for the urinary albumin:creatinine ratio. Clin Chem 1999;45:414–7. [4] Garg S, Hiar C, Pennington L, Osberg I, Hamilton R. A new reliable and rapid method for estimation of urinary albumin concentration. Diabetologica [Abstract] 1997;40(Suppl 1):2096. [5] Coresh J, Toto RD, Kirk KA, Whelton PK, Massry S, Jones C et al. Creatinine clearance as a measure of GFR in screens for the African American Study of Kidney Diseases and hypertension pilot study. Am J Kidney Dis 1998;32:32–42. [6] Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease study group. Ann Intern Med 1999;130:461–70. [7] Ruggenenti P, Gaspari F, Perna A, Remuzzi G. Cross-sectional longitudinal study of spot morning urine protein:creatinine ratio, 24-h urine excretion rate, glomerular filtration rate, and end-stage renal failure in chronic renal disease in patients without diabetes. Br Med J 1998;316:504–9. [8] Moore Jr. RR, Hirate-Dulas CA, Kasiske BL. Use of urine specific gravity to improve screening for albuminuria. Kidney Int 1997;52:240–3. [9] Mason HJ, Williams NR, Morgan MG, Stevenson AJ, Armitage S. Influence of biological and analytical variation on urine measurements for monitoring exposure to cadmium. Occup Environ Med 1998;55:132–7. [10] Lenter C. Body fluids, urine, nitrogenous substances. In: Lenter C, editor, Geigy Scientific Tables, West Caldwell NJ: Ciba-Geigy Corporation, 1981, p. 63. [11] Vestergaard P, Leverett R. Constancy of urinary creatinine excretion. J Lab Clin Med 1958;51:211–8. [12] Cockcroft DW, Gault MH. Prediction of the creatinine clearance from serum creatinine. Nephron 1976;16:311–41. [13] Corcuff JB, Tabarin A, Rashedi M, Duclos M, Roger P, Ducassou D. Overnight urinary free cortisol determination: a screening test for the diagnosis of Cushing’s syndrome. Clin Endocrinol (Oxford) 1998;48:503–8. [14] Bjorgaas M, Sagen E, Johnson H, Vik T, Sager G. Urinary excretion of catecholamines in hospitalized and non-hospitalized healthy children and adolescents. Scand J Clin Lab Invest 1998;58:339–46. [15] Pugia MJ, Lott JA, Kajimi J, Takaaki S, Sasaki M, Kuromoto K et al. Screening school children for albuminuria, proteinuria and occult blood with dipsticks. Clin Chem Lab Med 1999;37:149–57. [16] Pugia MJ, Lott JA, Luke KE, Shihabi ZK, Wians Jr. FH, Phillips L. Comparison of instrument-read dipsticks for albumin and creatinine in urine with visual results and quantitative methods. J Clin Lab Anal 1998;12:280–4. [17] Gowans EM, Fraser CG. Biological variation of serum and urine creatinine and creatinine clearance: ramifications for interpretation of results and patient care. Ann Clin Biochem 1988;25:259–63.

D. J. Newman et al. / Clinica Chimica Acta 294 (2000) 139 – 155

155

[18] Howey JE, Browning MC, Fraser CG. Biologic variation of urinary albumin: consequences for analysis, specimen collection, interpretation of results, and screening programs. Am J Kidney Dis 1989;13:35–7. [19] Ginsberg JM, Chang BS, Matarese RA, Garella S. Use of single voided urine samples to estimate quantitative proteinuria. New Engl J Med 1983;309:1543–6. [20] Poortmans JR, Engels MF, Sellier M, Leclercq R. Urine protein excretion and swimming events. Med Sci Sports Exercise 1991;7:831–5. [21] Poortmans JR, Vancalck B. Renal glomerular and tubular impairment during strenuous exercise in young women. Eur J Clin Invest 1978;8:175–8. [22] Robertshaw M, Cheung CK, Fairly I, Swaminathan R. Protein excretion after prolonged exercise. Ann Clin Biochem 1993;30:34–7. [23] Yaguchi H, Ishigooka M, Hayami S, Kobayashi T, Nakada T, Mitobe K. The effect of triathlon on urinary excretion of enzymes and proteins. Int Urol Nephrol 1998;30:107–12. [24] Armstrong LE, Maresh CM, Castellani JW, Mergeron MF, Kenefick RW, LaGasse KE et al. Urinary indices of hydration status. Int J Sport Nutr 1994;4:265–79.