Reference Intervals for 24-Hour and Random Urine Porphyrins*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 26, No. 4 Copyright 1996, Institute for Clinical Science, Inc. Reference Intervals for 24-Hour and Random Urine Porphyrins* KERN L. NUTTALL, M.D., Ph.D STEPHEN S. PINGREE, B.S. and EDWARD R. ASHWOOD, M.D. Department of Pathology University of Utah School of Medicine Salt Lake City, UT ABSTRACT Urine porphyrin analysis is an important part in evaluation of photosensitivity. Since porphyrin excretion is variable throughout the day, analysis is traditionally based on 24-hour collections. To facilitate the use of random specimens, as well as poorly collected 24-hour specimens, reference limits based on the porphyrin to creatinine ratio have been developed. Based on 1,171 adult specimens, it is estimated that the 95 percent reference limit (90 percent confidence interval) is ^3.9 ( ) ^mol/mol of creatinine for uroporphyrin and = 22 (19 34) (xmol/mol for coproporphyrin. These values apply to both 24-hour and random specimens, although random specimens show a higher degree of variability. Modest differences exist between males and females, but they are not significant given the degree of uncertainty in the confidence intervals. In terms of more traditional 24-hour units, reference limits correspond to = 37 (32 63) nmol/day for uroporphyrin and =s221 ( ) nmol/day for coproporphyrin. Introduction Urine porphyrin analysis can play an important part in the evaluation of photosensitive conditions. With reverse phase high performance liquid chromatography (HPLC), the pattern of abnormal porphyrin excretion associated with overt porphyria cutanea tarda (PCT) is often massive and highly distinctive (figure l ).1 The presence of isocoproporphyrin on the chromatogram is particularly characteristic of PCT. While some authors * Send reprint requests to: Dr. Kern Nuttall, c/o ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT believe that the category of PCT-type skin lesions should never be evaluated on the basis of urine porphyrin analysis alone,2 in practical terms urine analysis can support or exclude the diagnosis of PCT with a high degree of confidence.1 At the same time, it should also be appreciated that urine analysis is one part of a definitive evaluation, and the testing of additional specimen types is indicated when the clinical presentation is unusual or response to therapy is not prompt. Since porphyrin excretion is variable throughout the day, urine porphyrin analysis is traditionally based on 24-hour collections. The need for 24-hour collections causes a variety of problems dis /96/ $01.50 Institute for Clinical Science, Inc.

2 3 1 4 NUTTALL, PINGREE, AND ASHWOOD Time, min FIGURE 1. Representative urine porphyrin chromatograms from: (A) patient with overt porphyria cutanea tarda (PCT); (B) patient with successfully treated (sub-clinical) PCT; and (C) normal individual. Numbers next to peaks refer to the number of carboxyl groups in the porphyrin molecule (8 corresponds to uroporphyrin, 7, 6, and 5 to hepta-, hexa-, and penta-carboxylporphyrin, and 4 to coproporphyrin). The unlabeled peak between 5 and 6 corresponds to isocoproporphyrin. IS refers to mesoporphyrin, the internal standard. Early peaks are due to normally occurring fluorescent compounds in urine. tressingly familiar to most laboratorians. These problems are compounded in a clinical reference laboratory setting such as ours, where the specimens are collected at distant locations, and typically accompanied by little or no clinical information. It is not uncommon for even the sex of the patient to be unknown. The traditional reference limits used in this laboratory are based on an in-house study of 24-hour collections from laboratory employees and have been in place for some time.1 Although similar to reference ranges published elsewhere,3 it has long been suspected that a different approach to reference ranges would solve some commonly encountered problems. For example, although about 5 percent of patient specimens are expected to be identified as elevated, significantly more than expected fall outside the reference limits. Isolated elevations of coproporphyrin are particularly common.1 With traditional ranges, high volume specim ens almost inevitably appear abnormal, whereas low volumes have the opposite tendency. While it is possible to reject specimens that do not appear optimal, it is preferred to take a different approach based on our experience with random specimens. It is our belief that random urine can almost always provide clinically relevant information. The majority of random specimens show chromatograms that can exclude PCT with confidence (C in figure 1) or demonstrate chromatograms characteristic of PCT (A in figure 1). Although porphyrin excretion is variable, the variations tend to be moderate. In order to facilitate the use of random specimens, as well as to cope with poorly collected and processed 24-hour specimens, reference limits based on the porphyrin to creatinine ratio has been developed. Methods Urine porphyrins were analyzed by HPLC as described elsewhere.1,4 Briefly, filtered urine was loaded onto an automated HPLC system * and 200 jxl injected under instrument control onto a C18 reverse-phase column.t The sample was eluted with a methanol/phosphate gradient, porphyrins measured by fluorescencet and chromatograms printed under software control. Creatinine was determined on urine aliquots on a model 717 analyzerll using an automated kinetic Jaffe method. Selection of reference specimens was based on the indirect, random sampling strategy described by Solberg.5 The * Waters, Milford, MA t C5, Rainin Instrument Co., Woburn, MA t Fluorochrome detector, Varian Instrum ents, San Fernando, CA Millenium 2010, Waters, Milford, MA f Hitachi Instruments, San Jose, CA

3 population of study specimens consisted of all adult urine porphyrin results (1764 specimens) from January September, 1995 (table I). Exclusion criteria consisted of: (1) Specimens consistent with porphyrin disorders as will be described, (2) specimens with incomplete data (primarily unknown sex), and (3) dilute specimens (defined as <25 mg/dl creatinine). Those remaining were classified as reference specimens in table I. Additional exclusion criteria were applied to create an idealized 24-hour category. This exclusion criteria consisted of low total volume (<600 ml), high total volume (>2500 ml), low total creatinine (<600 mg/day), and high total creatinine (>2000 mg/day). TABLE I Exclusion Criteria for Study Population REFERENCE INTERVALS FOR PORPHYRINS Hour Random Total population Acute porphyria 13 2 Florid porphyria cutanea tarda Mild porphyria cutanea tarda 28 2 Incomplete data 26 8 Dilute Reference limits and confidence intervals were determined by the nonparametric method based on the rank number described by Solberg.5 Briefly, the sample population was ranked in ascending order of magnitude, and the 95 percent reference limit determined from the rank number equal to 0.95 (n + 1), where n was the number of specimens. Likewise, the 99 percent reference limits were calculated from the rank number equal to 0.99 (n + 1). Confidence intervals were taken from accompanying tables. Diagnosis of Porphyrin Disorders In our laboratory, porphyrin chromatograms are interpreted using the logic summarized in the flow diagram in figure 2. When the characteristic pattern is seen on the chromatogram (A in figure 1), the laboratory diagnosis of PCT is unambigu- Reference specimens* M F Low volume 58 - High volume 232 Low creatinine 77 - High creatinine 78 Reference specimens, idealizedb M 289 F a Total minus porphyrin disorders, those with incomplete data, and dilute specimens (<25 mg/dl creatinine). b Reference specimens having volumes between ml, and creatinines between mg/day. FlGURE 2. Flow diagram for the interpretation of urine porphyrins (abbreviations: PCT, porphyria cutanea tarda; c/w, compatible with; uro-, uroporphyrin; hepta-, heptacarboxy[porphyrin; copro-, coproporphyrin; and PBG, porphobilinogen).

4 3 1 6 NUTTALL, PINGREE, AND ASHWOOD ous,1 and porphyrins are present at concentrations which are capable of inducing overt cutaneous photosensitivity. As PCT is successfully treated, the porphyrin concentrations drop, leaving more modest elevations of uro- and heptacarboxyporphyrins (B in figure 1). The point at which a patient is no longer susceptible to photosensitivity depends on a variety of clinical factors, but the pattern of elevated uro- and heptacarboxyporphyrins is almost always associated with PCT, be it overt, subclinical, or latent. These values are interpreted by us as consistent with PCT, in contrast to the definitive diagnosis which also requires blood and fecal analysis. The lack of elevated porphyrins (C in figure 1) excludes PCT with a high degree of confidence. The acute neurologic porphyrias can also perturb the pattern of urine porphyrins.1,2 For this reason, whenever an unusual chromatographic pattern is present, an additional test for porphobilinogen (PBG) is added (if one has not already been requested). A common example of an unusual pattern is an uroporphyrin elevation in the absence of a similar elevation of heptacarboxyporphyrin. A normal PBG concentration excludes an episode of an acute porphyria with a high degree of certainty. Remaining elevations are typically moderate and non-diagnostic of porphyrin disorders. Results The exclusion criteria for defining reference specimens are listed in table I. Out of 1,764 total specimens received for porphyrin analysis over the 9 month study period, 15 (0.85 percent) were classified as diagnostic of an acute porphyria on the basis of a elevated urine PBG. (Of these, 8 were received with requests for PBG testing, and 7 were not.) An additional 126 (7.1 percent) were characteristic of overt PCT, and 30 (1.7 percent) were consistent with subclinical or latent PCT. Lack of patient information was used to exclude 34 specimens, primarily for lack of male/female designation. (Although 284 specimens (16 percent) were received without sex identification, in the majority of cases sex was extrapolated from the name with a reasonable degree of certainty.) Dilute specimens were excluded in 78 of hour specimens (5.4 percent) and for 27 of 324 random specimens (8.3 percent). The low est creatinine was 2 mg/dl (although suggestive of serum, this was a urine specimen). In order to investigate the effect of inadequate specimen collection on the study, additional exclusion criteria were applied to define a category of idealized reference specimens listed in table I. Out of 1, hour specimens, 445 (38 percent) have volume and creatin ine characteristics susp icious for improper collection. In terms of uroporphyrin (table II), the present study shows similar reference limits compared to the traditional limits for males and females. Although confidence intervals show significant overlap, male ranges are, in general, slightly higher than those for females. Since the number of specimens is largest in the 24-hour category (n = 1,174), the confidence interval is more narrowly defined for this group. There is a trend toward higher reference limits, starting with the idealized 24-hour specimens compared to the 24-hour category and to random specimens. Examining cumulative frequency distributions in figure 3 illustrates similar relationships. Random specimens show more variation than 24-hour specimens, even though random specimens merge with the 24-hour distribution at the upper end. In terms of coproporphyrin (table III), reference limits are significantly higher in the present study than traditional limits. Again, there is a trend toward higher reference limits starting with idealized

5 24-H ours, idealized REFERENCE INTERVALS FOR PORPHYRINS TABLE II Comparison of Uroporphyrin Reference Limits Traditional Present Studv3 < nmol/day < nmol/day < \imol/mol M ( ) 3.9 ( ) F ( ) 3.4 ( ) M /F 3 7 ( ) 3.6 ( ) 24-H ours M (41-84) 3.7 ( ) F ( ) 4.1 ( ) M /F 3 8 ( ) 3.9 ( )b Random 4.4 ( ) a Nonparametric 95 percent reference limit (90 percent confidence interval). b Recommended uroporphyrin reference limit. 24-hour specim ens compared to the 24-hour category and to random specimens. Random specim ens show the highest reference limit, although it is within the confidence interval of the 24- hour group. Discussion It is believed that a major reason for the differences seen in the present study compared to the more traditional reference limits (tables II and III) is primarily F i g u r e 3. C um ulative frequency distribution of uroporphyrin for 24-hr, id ea lized 24-hr, and random referen ce specimens. The error bar represents the 95 percent upper referen ce lim it with 90 percen t confidence limits for the 24-hr reference specimens Uroporphyrin, jimol/mol 6

6 3 1 8 NUTT ALL, PINGREE, AND ASHWOOD si 3 100% F i g u r e 4. Cum ula- "* 8 0 % tive frequency distribu- potion of coproporphyrin for 24-hr, id ea lized 24-hr, and random referen ce 6 0 % specimens. The error bar represents the 95 percent ^ upper referen ce lim it w ith 90 percent confi- g AC\OJ dence limits for the 24-hr ~ reference specimens Coproporphyrin, fimol/mol in the reference population selected. What has been labeled as traditional limits was an in-house study conducted some years ago on laboratory employees. These traditional limits are similar to those published elsewhere.3 Since laboratory employees are relatively healthy, constructing reference ranges from patient specimens specifically undergoing evaluation for porphyrin disorders is a more clinically appropriate population. Using the reference limits defined from this population of actual clinical specimens will result in considerably fewer isolated coproporphyrin results being labeled as abnormal. TABLE III Comparison of Coproporphyrin Reference Limits Traditional < nmol/day Present Study3 < nmol/day < nmol/mol 24-H ours, idealized M F M/F ( ) 175 ( ) 190 ( ) 22 ( ) 1 9 ( ) 20 ( ) 24-H ours M ( ) 21 (17-34) F ( ) 2 3 ( ) M/F 221 ( ) 2 2 ( )» Random 29 ( ) a Nonparametric 95 percent reference limit (90 percent confidence interval). b Recommended coproporphyrin reference limit.

7 REFERENCE INTERVALS FOR PORPHYRINS It was found that the indirect random sampling strategy described by Solberg5 was particularly suitable for our circumstances, since data collected during the routine operation of the laboratory could be used. A nonparametric method of data analysis was employed because it does not require the assumption of a Gaussian distribution among the reference specimens. Reference ranges for urine porphyrins have traditionally been divided into male and female ranges,1,3 and moderate sex differences clearly exist. However, in the present study the confidence intervals between males and females overlap, suggesting the differences are small compared to the uncertainty of defining reference limits. Sex specific ranges also complicate interpretation when sex is unknown, as occurred with 16 percent of the specimens in the present study. Such lack of basic information is common in reference laboratories where testing is often distant from the patient. It was concluded by us that the differences between males and females are so small as to contribute nothing practical to the diagnostic process in the clinical reference laboratory. The value of including confidence intervals speaks for itself, although it is not a common practice. It is suspected that if confidence intervals were included more often, less would be made of clinically insignificant differences. Exactly 38 percent (445/1171) of our 24-hour reference specimens has been labeled as suspicious for inadequate collection, although there is no way of confirming these suspicions. It is suspected that this figure actually underestimates the problem, since it does not include those specimens where the volume has been poorly estimated. The effect of excluding these suspicious specimens from the reference range is evident but not statistically significant. It does suggest that reference ranges based on ideal 24-hour collections do not reflect the specimens variability seen in actual practice. Defining the reference limits in terms of creatinine renders the effect of volume moot, enabling high and low volume specimens to be evaluated in more suitable terms. High volume specimens represent a category that are suspicious for processing reasons. Specimens >2,500 ml are typically collected in more than one container. (The largest volume in our study population was 8,500 ml.) The proper way to take a representative aliquot from specimens in multiple containers is to first pour the entire collection into a single large container, mix well, and remove an aliquot. It is suspected that this does not happen often in actual practice and that aliquots from large volumes are seldom representative of the entire collection. Concerns about total volumes and creatinines involve issues about the way data are manipulated and interpreted, whereas dilute specimens represent a more fundamental problem. D ilu te specimens limit the sensitivity of the analytic process. At some point, urine becomes too dilute to be an accurate indicator of patient status no matter how the analytic data is interpreted. Greater than 25 mg/dl creatinine has been as our cutoff, although this value is based on a subjective opinion of what constitutes sign ifica n t d ilu tio n. W hen a d ilu te specimen is received, it is tested in the usual fashion, but a footnote is added suggesting that the specimen may be too dilute for accurate testing. When the dilute specimens in table I are included in the reference specimens, the resulting reference limits are not altered substantially (data not shown). However, this category has been excluded to emphasize the fundamental analytic problem s caused by dilute specimens. D e f i n i n g P o r p h y r i n E l e v a t i o n s It is recommended by the current authors to use the 95 percent reference

8 3 2 0 NUTTALL, PINGREE, AND ASHWOOD limits (90 percent confidence intervals) for uroporphyrin of = 3.9 (3.5 to 5.7) xmol/mol creatinine (table II) and for coproporphyrin of =s22 (19 to 34) xmol/ mol creatinine (table III). This is the reference group that contains the largest number of specimens and, therefore, has the most narrowly defined confidence intervals. The fact that the confidence intervals are still relatively broad should emphasize that these limits are not absolute, and should be interpreted within an appropriate context. The main advantage to using the porphyrin to creatinine ratio is that the reference limits can be applied to random specimens. Such reference limits may also compensate for poorly collected 24-hour specim ens, especially those where the volumes are extreme. It is recognized that using a simple creatinine ratio is not ideal.6,7,8 The urinary excretion of many substances is not independent of creatinine, and other more complicated methods have been suggested to compensate in a more precise fashion. However, since the confidence intervals in the present study indicate that there is substantial uncertainty to the limits, the simple creatinine ratio is adequate for our purposes. This distinction between slight, moderate, and massive porphyrin excretion is a somewhat arbitrary distinction for the clinical laboratory, but one that contains important information for the diagnosis of photosensitive conditions. Slight elevations are defined as up to 5 times the reference limit, moderate elevations as between 5 to 10 times the reference interval, and massive elevations as >10 times. In those patients excreting massive porphyrin concentrations, photosensitive conditions can be attributed to the elevated porphyrin concentrations. Likewise, in patients with slight elevations, photosensitive conditions are much more likely to be due to other events such as drug reactions. Biochemical remission in PCT has been defined by Kalb et al9 as a porphyrin excretion of less than 400 (xg/day. (Biochemical remission often follows clinical remission with a reduction of blister formation and skin fragility by months or even years.) Changing units, this is equivalent to a uroporphyrin excretion of 480 nmol/day, or about 10 times the 95 percent reference limit for 24-hour collections listed in table II. Misclassifying an individual having slightly elevated porphyrins as normal carries relatively little risk, since clinically significant elevations are much higher. However, misclassifying a high normal individual as elevated is considerably more likely to have deleterious effects. The most obvious impact is the cost of additional studies generated in response to findings incorrectly identified as abnormal. D i a g n o s t i c P r o b l e m s The definitive diagnosis of porphyrin disorders often requires much more than urine porphyrin analysis. Some authors believe that the category of PCT-type skin lesions should never be evaluated on the basis of urine porphyrin analysis alone, and advocate the simultaneous analysis of blood and fecal porphyrins.2 Such tests may be necessary to distinguish PCT absolutely from entities such as variegate porphyria, the adult form of congenital erythropoietic porphyria, and mixed porphyrin disorders, although these conditions are considerably more infrequent than PCT. In practical terms, urine analysis can support or exclude the diagnosis of PCT with a high degree of confidence. However, it should also be appreciated that additional testing may be indicated particularly when the clinical presentation is usual, or w hen response to therapy is not prompt. It is not uncommon for the laboratory to discover that urine porphyrins were ordered by mistake instead of urine porphobilinogen (PBG), and that the physician was concerned about excluding an

9 acute porphyria like acute intermittent porphyria (AIP). Acute porphyrias were identified in the present study on 15 occasions, and 7 of these 15 did not have the appropriate PBG test ordered. This is due to a number of factors including the closely related names and the unfamiliarity of many physicians with porphyrin disorders. In our experience, the majority of acute porphyrias cause significant perturbations in urine porphyrins, although several instances where this is not the case have been seen by us. Elevated PBG can undergo spontaneous polymerization to form the uroporphyrin type-i isomer,1,2 particularly as specimens age. This can cause either an isolated elevation of uroporphyrin or a massive elevation of uroporphyrin with only a slight elevation of heptacarboxylporphyrin. Whenever an atypical or unusual pattern of porphyrins is seen on the chromatogram, an additional test for PBG is indicated. However, it has also been found that the majority of specimens containing isolated moderately-elevated uroporphyrins are not due to acute porphyrias and are not diagnostic porphyrin disorders. In general, the finding of an isolated porphyrin elevation is reason to scrutin ize the chrom atogram carefu lly. Although the combination of HPLC with fluorescence detection is highly specific, it is not absolutely definitive, and drug interferences are known.1,10 Isolated coproporphyrin elevations are relatively common and nondiagnostic, often being secondary to diverse conditions like diet and liver disease. Although use of the coproporphyrin reference limit defined in the present study will reduce the number of results labeled as elevated, 5 percent of nondiagnostic specimens are still expected to be above the reference limit. Conclusions Urine reference lim its have been defined in terms of the porphyrin to creatinine ratio in order to facilitate the use REFERENCE INTERVALS FOR PORPHYRINS of random specimens and poorly collected 24-hour specim ens. Recom mended 95 percent reference limits (90 percent confidence intervals) are = 3.9 (3.5 to 5.7) (xmol/mol of creatinine for uroporphyrin (table II), and = 22 (19 to 34) (xmol/mol for coproporphyrin (table III). These limits are applicable to both 24-hour and random specimens, although random specimens show increasing variability. There is a significant degree of uncertainty as seen from the confidence intervals. In terms of more traditional 24-hour units, these limits correspond to = 37 (32 to 63) nmol/day for uroporphyrin, and ^221 (195 to 320) nmol/day for coproporphyrin. Although similar for uroporphyrin, the limits for coproporphyrin are higher in the present study compared to previously published ranges.1,3 Differences between males and females are small and do not contribute significantly to practical diagnostic issues. Slight elevations have been defined arbitrarily as 1 to 5 times the reference limits, moderate elevations as 5 to 10 times, and massive elevations as >10 times. Susceptibility to photosensitivity depends on a variety of clin ical circum stances, but can be expected with massive elevations. References 1. Nuttall KL. Porphyrins and disorders of porphyrin metabolism. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry, 2nd ed. Philadelphia: WB Saunders, 1994: Elder GH, Smith SG, Smyth SJ. Laboratory investigation of the porphyrias. Ann Clin Biochem 1990;27: Tietz NW, ed. Clinical Guide of Laboratory Tests, 3rd ed. Philadelphia: Saunders, 1995: Ford RE, Ou CN, E llefson RD. L iquidchromatographic analysis for urinary porphyrins. Clin Chem 1981;27: Solberg HE. Establishment and use of reference values. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry, 2nd ed. Philadelphia: WB Saunders, 1994: Cole M, Craft AW, Parker L, Bell S, Seviour JA, M cg ill AC, D ale G. Urinary creatin in e adjusted reference ranges for homovanillic and

10 3 22 NUTTALL, PINGREE, AND ASHWOOD vanillylmandelic acid in children and adults. Clin Chim Acta 1995;236: Fried PA, Perkins SL, Watkinson B, McCartney JS. Association betw een creatinine-adjusted and unadjusted urine continine values in children and the mother s report of exposure to environmental tobacco smoke. Clin Biochem 1995;28: Greenber GN, Levine RJ. Urinary creatinine excretion is not stable: a new method for assessing urinary toxic substance concentrations. J Occup Med 1989;31: Kalb RE, Grossman ME, Poh-Fitzpatrick MB. Correlation of serum and urinary porphyrin levels in porphyria cutanea tarda. Arch Dermatol 1985;121: Van Pelt J, Verheesen PE, van Oosterhout AGM. Interference of dipyridamole in the analysis of porphyrins by HPLC. Ann Clin Biochem 1992;29:347-8.