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Urinary Porphyrin Excretion in Normal Children and Adults
Urinary Porphyrin Excretion in Normal Children and Adults Kenneth E. Bloom, MD, Edith F. Zaider, MS, Louis J. Morledge, MD, and Maureen B. Poh-Fitzpatrick, MD • The relationship of random urinary porphyrin and creatinine values as functions of age and sex was examined in a normal population. Total urinary porphyrin was measured by a solvent extraction technique, while urinary creatinine was evaluated by an alkaline picrate method. Random urine specimens from 120 healthy patients (81 children and 39 adults) were evaluated. In both pediatric and adult populations, a strong correlation was found between urinary concentrations of porphyrin and creatinine (r = 0.7, P < 0.0001). Urinary porphyrin excretion in "mol/mol creatinine ("g/ g) was inversely related to both age (r = -0.59, P < 0.0001) and weight (r = -0.61, P < 0.0001) until approximately 9 years of age or 30 kg. Urinary porphyrin excretion in children 9 to 18 years of age was lower than that of younger children (P < 0.0001) and approached adult values. Sex was not found to be a factor until 9 to 18 years of age, when females had higher urinary creatinine concentrations (P < 0.05), but lower urinary porphyrin excretions (P < 0.05) than similarly aged males. The converse was observed when similar values of adult women were compared with those of adult men. Men also had higher urinary porphyrin concentrations than women (P < 0.01). Men had increased urinary creatinine concentration (P < 0.05) and decreased porphyrin excretion ratios (P < 0.05) when compared with males 9 to 18 years of age. Women had significantly lower urinary creatinine (P < 0.001) and porphyrin (P < 0.001) concentrations than females 9 to 18 years of age. Quantitative analysis of porphyrin and creatinine concentrations in random urinary specimens can provide diagnostically informative assessments of porphyrinuria, although effects of age, weight, and sex and of diurnal variation of porphyrin excretion must be considered. © 1991 by the National Kidney Foundation, Inc. INDEX WORDS: Coproporphyrin; creatinine; porphyrin; porphyrinuria.
U
RINARY PORPHYRIN excretion may be abnormally elevated in many diseases. 1,2 These include porphyrias,3,4 lead poisoning, S,6 liver disease 7 (ie, hepatitis, cholestasis, cirrhosis, tumors,2 Rotor syndrome,8 some cases of Dubin Johnson syndrome,9 Alagille syndrome,1O exposure to hepatotoxic or porphyrinogenic substances (ie, polyhalogenated aromatics I 1,12), disorders of hemoglobin synthesis (ie, thalassemia I 3), chloromas, 2 acute pyogenic infections, 2 poliomyelitis,14 and transiently in meconium aspiration of the newborn. ls Evaluation of patients with disorders of porphyrin metabolism often requires porphyrin analyses of urine, stool, serum, and erythrocytes. 4 Urinary porphyrin levels are traditionally expressed in adults as ~g/dL or per 24-hour volume. 16-19 As 24-hour urine collections are difficult in children, pediatric values have been expressed either per deciliter or per gram creatinine, but normal reference values have not been uniformly determined for all pediatric age groups. The purpose of this study was to determine the relationship of random urinary determinations of porphyrin and creatinine as functions of age and sex in normal children and adults. MATERIALS AND METHODS
Specimens Random urine specimens were collected from clinically normal pediatric and adult patients during routine daytime
ambulatory visits, with no restrictions on fluid or dietary intake. None had known disorders of porphyrin metabolism, anemia, lead intoxication, exposure to exogenous hormones or to hepatotoxic or porphyrinogenic substances, gastrointestinal symptoms, or concurrent infection. All children weighed within 2 SD of the mean for age using a standard pediatric growth chart. 20 Pediatric ages varied from 9 months to 17 years 9 months, and adult ages ranged from 22 years to 49 years when approximated to the nearest 3-month interval. Sex was identified for 76 pediatric (32 females and 46 males) and for 39 adult (14 women and 25 men) samples. Specimens were eliminated if pyuria or hematuria was found on routine urinalysis. Liver chemistries were not deemed necessary in this clinically normal population. All samples were protected from fluorescent light, refrigerated, and evaluated within 10 days or frozen at 0 0c.
Urinary Porphyrin Determinations Urinary total porphyrins were measured by adapting the free erythrocyte porphyrin fluorometric assay described by Poh-Fitzpatrick et al21 as follows: 200 "L of urine was added From the Department o/Dermatology, New York Medical College, Valhalla, NY. Supported in part by Research Grant No. R01 AR 18549 from the National Institute 0/ Arthritis, Musculoskeletal and Skin Disease, Department 0/ Health and Human Services. K.E.B. is currently at the Department 0/Dermatology, University 0/ Minnesota, Minneapolis, MN; L.J.M. is currently at the Lenox Hill Hospital, New York, NY. Address reprint requests to Maureen B. Poh-Fitzpatrick, MD, Acting Chairman, Department o/Dermatology, New York Medical College, Vosburgh Pavilion, 2nd Floor, Valhalla, NY 10595. © 1991 by the National Kidney Foundation, Inc. 0272-6386/91/1804-0008$3.00/0
American Journal of Kidney Diseases, Vol XVIII, No 4 (October), 1991: pp 483-489
483
484
BLOOM ET AL 765(50)',------------------,
to 0.3 mL of 5% infusorial earth (Celite; Fisher Scientific, Springfield, NI) in a pH 7.4 phosphate-buffered saline solution. Calculations necessary to obtain the quantitative porphyrin values were similarly modified:
'E ~ 612(40} >'..J
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Total urinary porphyrin nmol/L =
[L..J
C, 2.9 100 F. X - X - X F, 0.2 1.08
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306(20}
::::>
153(10)
where F. is fluorescence of sample; C" concentration of coproporphyrin standard (0.05 JIg/mL); F" fluorescence of standard; 2.9, final volume ofHCI phase (increased from original 2 mL); 0.2, original volume of urine; 100, conversion factor to 100 mL of extract; and 1.08, conversion factor for the enhancement of porphyrin fluorescence by ethyl acetate saturation of 1.5N HCI; and 15.3, conversion factor from JIg/ dL to nmol/L. This gives total urinary porphyrin measured as nanomoles (nmol) coproporphyrin per liter, understanding that approximately 70% to 80% of total urinary porphyrins in normal individuals are coproporphyrins (I JIg/dL = 15.3 nmol/L).
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Urinary Creatinine Determinations
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Urinary creatinine was measured using the Technicon RA System method (Technicon Instruments Corp., Tarrytown, NYf2 based on the Jaffe alkaline picrate reaction.23 The method was automated by Chasson et aV' and optimized by Rossignol et al. 25 Urine samples were diluted fivefold for determinations and results were expressed in micromoles per liter (I mg/dL = 88.4 JImoI/L).
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Fig 1. Total urinary porphyrin as a function of (A) weight and (8) age in pediatric specimens.
Statistical Analysis
to age in children. The direct relationship of values of urinary porphyrin and creatinine in both pediatric and adult specimens is shown in Fig 2. Urinary porphyrin excretion expressed as ~mol/ mol creatinine (or ~g/g) is inversely related to both weight and age in younger children (Fig 3A and B) (I ~gcoproporphyrin/g creatinine = 0.173
Statistical analysis was performed using the Statistical Analysis System programs of SAS Institute, Cary, NC, for linear regression (General Linear Model) and correlation.
RESULTS
Figures 1A and B show total urinary porphyrin concentration (nmol/dL) related to weight and
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• age ( 18 years fr = 0.72, P ( 0.0001] age.t 18 years r = 0 .77, P ( 0 0001] .
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Fig 2. Urinary porphyrin as a function of urinary creatinine in pediatric and adult specimens. The solid and dashed lines represent regression lines in the pediatric and adult populations, respectively.
485
URINARY PORPHYRIN EXCRETION c:
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JLmol/mol). Linear regression analysis was performed on the pediatric data separated into two groups at the point where total urinary porphyrin excretion becomes independent of weight or age (30 kg and 9 years). The urinary porphyrin excretion (JLmol porphyrin/mol creatinine or JLg/g) of patients 9 to 18 years of age was lower than values of younger children (P < 0.0001), but did not significantly differ (P = 0.63) from adult values when not segregated by sex. Pearson correlation coefficients (r) and significance values (P) of the relationships in the pediatric patients of (1) urinary porphyrin concentration and urinary porphyrin excretion to age and weight, (2) urinary creatinine to urinary porphyrins, and (3) creatinine to age and weight are displayed in Table 1. Urinary porphyrins, creatinine, and their excretion ratio are displayed with respect to sex in Figs 4a, b, and c. Dummy variable analysis (Table 2) did not show sex as a statistically significant factor in patients less than 9 years of age. However in the 9- to 18-year-old patients, females had higher urinary creatinine and porphyrin concentrations, but lower excretion of porphyrin per gram creatinine than similarly aged males. Adult men had higher urinary creatinine and porphyrin
Table 1. Correlation Coefficients and Levels of Significance of Measured Variables in Pediatric Patients
Dependent Variable
Urinary creatinine mg/dL
Urinary porphyrin
~g/dL
(~mol/L)
(nmol/L)
Urinary porphyrin excretion creatinine (J'moljmol)
~g/g
* Pearson correlation coefficient.
t P value not significant at 0.05 level.
Independent Variable
No. of Patients
Urinary porphyrin ~g/dL (nmoIfL) Weight <30 kg ;;0,,30 kg Age <9 yr 9-18 yr Weight <30 kg ;;0,,30 kg Age <9 yr 9-18 yr Weight <30 kg ,,;;30 kg Age <9 yr 9-18 yr
r Value"
PValuet
81
0.72
<0.0001
48 33
0.45 0.10
<0.01 0.57t
50 31 81 48 33 81 50 31
0.49 0.15 0.19 0.11 0.01 0.23 0.17 -0.07
<0.001 0.43 0.09t 0.46t 0.95t <0.05 0.25t 0.72t
48 33
-0.59 -0.12
<0.0001 0.49t
48 31
-0.61 -0.30
<0.0001 0.10t
486
BLOOM ET AL
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(yeors) Fig 4. The effect of age and sex on (A) urinary porphyrin concentration (nmol/L or ",g/dL), (8) urinary creatinine concentration (nmol/L or ",g/dL), and (C) urinary porphyrin excretion (",mol/mol or ",g/g creatinine). All values are expressed as ±SE. D, Males; ~, females.
concentrations but lower porphyrin excretion than adult women. After segregating the data by sex, men had significant increases in urinary creatinine concentration and decreased porphyrin excretion ratios when compared with males 9 to 18 years of age (Table 3). When compared with females 9 to 18 years of age, the higher urinary porphyrin excretion of adult women approached significance (P = 0.05), while the values of their urinary creatinine and porphyrin concentrations were significantly lower (P < 0.00 I for both). DISCUSSION
In previous studies, Hsia and Page found that 24-hour coproporphyrin excretion correlated
better with weight than with age and body surface area in 6- to 16-year-old boys. 1Wolkoff and Arias measured creatinine and coproporphyrin in random urine specimens of 23 premature infants and calculated daily excretion of coproporphyrin by assuming a constant creatinine excretion of 8 mg/kg/24 h. 26 Rocchi et al found healthy infants to have a to-fold decrease in J-Lg coproporphyrin/ g creatinine during the first to days oflife. 27 Other studies indicate that creatinine increases with postnatal age. 28 •29 However, these studies do not provide a uniform assessment of the ranges of normal urinary porphyrin output in children of both sexes and all ages. In comparing our values with those of studies in which high performance liquid chromatography (HPLC) and/or absorption spectrophotometry were used to quantify urinary porphyrins, it should be noted that such methods often yield lesser quantities than those determined by solvent extraction and/or fluorometry.3o Using HPLC, Poh-Fitzpatrick et al found the mean coproporphyrin fraction of total urinary porphyrins in normal children to be at least 70%, with the majority as isomer m.1O We quantified total urinary porphyrins in these normal subjects as coproporphyrin in light of that data, and of data of Enriquez de Salamanca et al placing the mean coproporphyrin fraction of total urinary porphyrin in normal adults near 80%.16 Thus, in comparing our study with any study in which isolated coproporphyrin was quantified, our values would also tend to be higher. The increased concentration values of urinary porphyrin found in adult men are consistent with the higher quantitative coproporphyrinuria or total porphyrinuria found in males by previous investigators. 16,31 The higher concentration of urinary creatinine in men versus women results in lower porphyrin excretion values for men. In the 9- to 18-year-old group, females had higher concentrations of both porphyrin and creatinine, yet lower porphyrin excretion due to the greater magnitude of difference in the creatinine levels. Disparity in urinary creatinine concentrations between males and females in both older age groups probably reflects differences in body mass. Body mass may also be a factor contributing to the similar pattern seen when comparing urinary porphyrin concentrations in males versus females (Fig 4A).
487
URINARY PORPHYRIN EXCRETION Table 2. Effects of Gender and Age Sex Dependent Variable
Urinary creatinine (mg/dL)
Urinary coproporphyrin (/Lg/dL) Urinary prophyrin excretion (/Lg/g creatinine)
Age (yr)
s:9 9-18 >18 s:9 9-18 >18 s:9 9-18 >18
Male (n)t
77.6± 120.4 ± 174.8 ± 13.9 ± 16.8 ± 18.0 ± 209.5 ± 142.6 ± 110.3 ±
, Female (n)t
8.5 (28) 14.8 (16) 15.6 (26) 1.4 (28) 2.1 (16) 1.7 (26) 17.8 (28) 11 .1 (16) 7.7 (26)
89.0 ± 191.5 ± 76.2 ± 14.8 ± 20.2± 9.7 ± 195.5 ± 111.5 ± 142.4 ±
Value:!:
14.3 (19) 24.0 (13) 10.9 (13) 2.0 (19) 2.1 (13) 1.0 (13) 19.1 (19) 9.1 (13) 12.0 (13)
0.11 0 .45 0.57 0.04 0.21 0.49 0 .08 0.37 0.36
PValue§
0.47§ <0.05 <0.001 0.78§ 0.26§ <0.01 0.60§ <0.05 <0.05
, Values are expressed as mean ± SE. "n" denotes sample size. :j: Pearson correlation coefficient. § P value not significant at 0 .05 level.
t
Sanguinetti et al showed the effect of creatinine correction in coproporphyrinuria of workers exposed to lead. 33 Since diurnal variation in urinary porphyrin excretion has been observed in several independent studies, the recommendation that complete 24-hour specimens should still be collected whenever possible seems justified. However, in children or others unable to cooperate, measurement of total porphyrin excretion related to creatinine in random specimens obtained in midafternoon should not fail to detect abnormal coproporphyrinuria, if compared with normal values corresponding to the age, weight, and sex of the individual. Diurnal variation in urinary uroporphyrin and heptacarboxyporphyrin excretion inverse to that of coproporphyrin, with nadirs in the afternoon and higher levels from midnight to noon, was found by Martasek et al in 10 patients with porphyria cutanea tarda. 36 However, in the uropor-
Whether urinary excretion data obtained from random specimens are as useful as those from complete 24-hour specimens depends on the extent of diurnal variation in the metabolites of interest. Circadian rhythm in urinary coproporphyrin excretion, lower during night and morning and maximal in afternoon hours, has been observed in normal adult men 32 and in men with pathologic porphyrinuria,33-34 and can be speculated to result from changes in urinary pH35 or glomerular filtration, reabsorption, or secretion of porphyrins while supine. The values we report herein were from random daytime specimens collected with no attempt to control pH by dietary measures. Since urinary creatinine excretion also decreases during the night and morning and peaks in the afternoon,32,35 expressing porphyrin excretion per unit creatinine would tend to correct diurnal variance, but with greater efficiency in normal or only mildly elevated porphyrinuria.
Table 3. Comparison of Adults and Children (9-18 Years) of Same Gender Dependent Variable
Urinary creatinine mmol/L (mg/dL) Urinary porphyrin nmol/L (!Lg/dL) Urinary porphyrin excretion /Lmol/mol creatinine (!Lg/g)
Sex
, Value'
PValuet
Males Females Males Females Males Females
0.35 -0.67 0 .64 -0.67 -0.36 0.39
<0.05 <0.001 0.08' <0.001 <0.05 0.05t
, Pearson correlation coefficient with positivie values signify increased values of variable in adult population.
t P value not significant at 0.05 level.
488
BLOOM ET AL
phyrinuria in porphyrias occurring in children (porphyria cutanea tarda, congenital erythropoietic porphyria, hepatoerythropoietic porphyria, or other rare homozygous porphyric states), from whom collecting complete 24-hour specimens may be virtually impossible in an ambulatory setting, total porphyrin output is often so high that a falsely normal result by our method would be unlikely, even in a random specimen obtained at a circadian nadir. In such cases, absolute values for total urinary porphyrin as calculated in coproporphyrin units by our simple, rapid, and relatively inexpensive technique would be somewhat in error; however, the amounts of other urinary porphyrins excreted by patients with var-
ious porphyrias are frequently at least an order of magnitude greater than the total porphyrin output of normal adults or children, and would still yield a clearly abnormal result. If abnormal total porphyrinuria is found in random or 24hour specimens, more sophisticated fractionation methods should then be used to determine the relative amounts of porphyrins of different carboxyl number and isomer partition to aid in the determination of the precise diagnosis. ACKNOWLEDGMENT The authors thank Dr Valery Morris and Dr Robert Berk for their help in obtaining clinical specimens, and Jeanine Carr and Dr Gregory Almond for their technical assistance.
REFERENCES I. Hsia DY-Y, Page M: Coproporphyrin studies in children. I. urinary coproporphyrin excretion in normal children. Proc Soc Exp Bioi Med 85:86-88, 1954 2. Ford RE, Ou CN, Ellefson RD: Liquid-chromatographic analysis for urinary porphyrins. Clin Chern 29:527-530, 1981 3. Poh-Fitzpatrick MB, Ramsay CA, Frain-Bell W, et al: Photodermatosis in infants and children. Pediatr Dermatol 5:189-200, 1988 4. Poh-Fitzpatrick MB: Laboratory testing in the porphyrias. Int J Dermatol 18:452-460, 1979 5. Chisolm n, Barrett MB, Mellits ED: Dose-effect and dose-response relationships for lead in children. J Pediatr 87: 1152-1160, 1985 6. Piomelli S: A micromethod for free erythrocyte porphyrins: The FEP test. J Lab Clin Med 81 :932-940, 1973 7. Ostrowski J, Kosecki P, Martynska M, et al: Urinary porphyrins in liver disease. Scand J Gastroenterol 19:862-866, 1984 8. Wolkoff AW, Wolpert E, Pascasio FN, et al: Rotors syndrome: A distinct inheritable pathophysiologic entity. Am J Med 60:173-179, 1976 9. Wolkoff AW, Cohen LE, Arias 1M: Inheritance of the Dubin-Johnson Syndrome. N Eng! J Med 288: 113-117, 1973 10. Poh-Fitzpatrick MB, Zaider E, Sciales C, et al: Cutaneous photosensitivity and coproporphyrin abnormalities in Alagille syndrome. Gastroenterology 99:831-835, 1990 II. Gladen BC, Rogan WJ, Ragan NB, et al: Urinary porphyrins in children exposed transplacentally to polyhalogenated aromatics in Taiwan. Arch Environ Health 43:54-58, 1988 12. Cripps DJ, Peters HA, Gocmen A, et al: Porphyria turcica due to hexachlorobenzene: A 20 to 30 year follow up study on 204 patients. Br J Dermatol III :413-422, 1984 13. Lyberatos M, Papadopoulos N, Papasteriadis E, et al: Urine porphyrins and their percursors in homozygous P-thalassaemia. Acta Haematol 54:95-97, 1975 14. Watson CJ, Schulze W, Hawkinson Y, et al: Copro-
porphyrinuria (type III) in acute poliomyelitis. Proc Soc Exp Bioi Med 64:73-78, 1947 15. Francoual J, Lindenbaum A, Dehan M, et al: Coproporphyrin in urine of newborns with meconium aspiration syndrome. Clin Chern 29:2054-2056, 1983 16. Enriquez de Salamanca R, Pena ML, Chinarro A, et al: Quantitative and qualitative porphyrin excretion in normal subjects. Br J Biochem 14:251-254, 1982 17. Fernandez AA, Henry RJ, Goldenberg H: Assay of urinary porphyrins. Evaluation of extraction methods and choice of instrumentation. Clin Chern 12:463-474, 1966 18. Talman EL: Standard Methods in Clinical Chemistry, vol 2. San Diego, CA, Academic, 1958, pp 137-147 19. Sobel C, Cano C, Thiers RE: Separation and quantitation of coproporphyrin and uroporphyrin in urine. Clin Chern 20: 1397-1402, 1974 20. Green MG (ed): The Harriet Lane Handbook (Johns Hopkins Hospital) (ed 12). Chicago, IL, Year Book, 1991, pp 353-360 21 . Poh-Fitzpatrick MB, Piomelli S, Young P, et al: Rapid quantitative assay for erythrocyte porphyrins. Rapid quantitative microfluorometric assay applicable to the diagnosis of erythropoietic protoporphyria. Arch Dermatol 110:225-230, 1984 22. Technicon Method No. SM4-014IK86. Creatinine. Technicon Instruments Corp., Tarrytown, NY, October 1986 23. Jaffe MZ: Concerning the precipitate produced in normal urine by picric acid and a new reaction of creatinine. Z Physiol Chern 10:391-400, 1986 24. Chasson AI, Grady HJ, Stanley MA: Determinations of creatinine measurement by means of automatic chemical analysis. Am J Clin Pathol 35:83-88, 1961 25. Rossignol B, Rossignol D, PetitClerc C: Improvement of creatinine measurement on RA-IOOO. Clin Biochem 17: 2034, 1984 (abstr) 26. Wolkoff AW, Arias 1M: Coproporphyrin excretion in amniotic fluid and urine from premature infants: A possible maturation defect. Pediatr Res 8:591-593, 1974 27. Rocchi E, Balli F, Gibertini P, et al: Coproporphyrin excretion in healthy newborn babies. J Pediatr Gastroenterol Nutr 3:402-407, 1984
URINARY PORPHYRIN EXCRETION
28. Trompeter LS, aI-Dahhan J, Haycock GB, et aI: Normal values for plasma creatinine concentration related to maturity in normal term and preterm infants. Int J Pediatr Nephrol 4: 145-148, 1983 29. Ross B, Cowett RM, Oh W: Renal functions of low birth weight infants during the first two months oflife. Pediatr Res II : 1162-1164, 1977 30. Gaetani E, Laureri CF, Vitto M: High performance liquid chromatographic differentiation of urinary free porphyrins. J Chromatogr 231 :425-432, 1982 31 . Doss M, Schmidt A: Rapid determination of urinary total porphyrins by ion exchange chromatography. Z K1in Chim Biochem 9:415-418, 1971 32. Araki S, Murata K, Yokoyama K, et a1: Circadian rhythms in the urinary excretion of metals and organic sub-
489 stances in "healthy" men. Arch Environ Health 38:360-366, 1983 33. Sanguinetti F, Dompe M, Mantovani S: Circadian rhythms in the excretion of coproporphyrin and deltaaminolevulinic acid. Ann 1st Super Sanita 14:601-606, 1978 34. Martasek P, Jirsa P, Kordac V: Role of the kidneys in porphyrias. Nephron 32:277-278, 1982 35. Kanabrocki EL, Snedeker PW, Zieher SJ, et aI: Circadian characteristics of dialyzable and non-dialyzable human urinary electrolytes, trace elements and total solids. Chronobiol Int 5:175-184,1988 36. Martasek P, Jirsa M, Kordac V: Diurnal rhythm of excretion of various types of porphyrins in symptomatic hepatic porphyria. Cas Lek Cesk 120:1255-1257, 1981
U
RINARY PORPHYRIN excretion may be abnormally elevated in many diseases. 1,2 These include porphyrias,3,4 lead poisoning, S,6 liver disease 7 (ie, hepatitis, cholestasis, cirrhosis, tumors,2 Rotor syndrome,8 some cases of Dubin Johnson syndrome,9 Alagille syndrome,1O exposure to hepatotoxic or porphyrinogenic substances (ie, polyhalogenated aromatics I 1,12), disorders of hemoglobin synthesis (ie, thalassemia I 3), chloromas, 2 acute pyogenic infections, 2 poliomyelitis,14 and transiently in meconium aspiration of the newborn. ls Evaluation of patients with disorders of porphyrin metabolism often requires porphyrin analyses of urine, stool, serum, and erythrocytes. 4 Urinary porphyrin levels are traditionally expressed in adults as ~g/dL or per 24-hour volume. 16-19 As 24-hour urine collections are difficult in children, pediatric values have been expressed either per deciliter or per gram creatinine, but normal reference values have not been uniformly determined for all pediatric age groups. The purpose of this study was to determine the relationship of random urinary determinations of porphyrin and creatinine as functions of age and sex in normal children and adults. MATERIALS AND METHODS
Specimens Random urine specimens were collected from clinically normal pediatric and adult patients during routine daytime
ambulatory visits, with no restrictions on fluid or dietary intake. None had known disorders of porphyrin metabolism, anemia, lead intoxication, exposure to exogenous hormones or to hepatotoxic or porphyrinogenic substances, gastrointestinal symptoms, or concurrent infection. All children weighed within 2 SD of the mean for age using a standard pediatric growth chart. 20 Pediatric ages varied from 9 months to 17 years 9 months, and adult ages ranged from 22 years to 49 years when approximated to the nearest 3-month interval. Sex was identified for 76 pediatric (32 females and 46 males) and for 39 adult (14 women and 25 men) samples. Specimens were eliminated if pyuria or hematuria was found on routine urinalysis. Liver chemistries were not deemed necessary in this clinically normal population. All samples were protected from fluorescent light, refrigerated, and evaluated within 10 days or frozen at 0 0c.
Urinary Porphyrin Determinations Urinary total porphyrins were measured by adapting the free erythrocyte porphyrin fluorometric assay described by Poh-Fitzpatrick et al21 as follows: 200 "L of urine was added From the Department o/Dermatology, New York Medical College, Valhalla, NY. Supported in part by Research Grant No. R01 AR 18549 from the National Institute 0/ Arthritis, Musculoskeletal and Skin Disease, Department 0/ Health and Human Services. K.E.B. is currently at the Department 0/Dermatology, University 0/ Minnesota, Minneapolis, MN; L.J.M. is currently at the Lenox Hill Hospital, New York, NY. Address reprint requests to Maureen B. Poh-Fitzpatrick, MD, Acting Chairman, Department o/Dermatology, New York Medical College, Vosburgh Pavilion, 2nd Floor, Valhalla, NY 10595. © 1991 by the National Kidney Foundation, Inc. 0272-6386/91/1804-0008$3.00/0
American Journal of Kidney Diseases, Vol XVIII, No 4 (October), 1991: pp 483-489
483
484
BLOOM ET AL 765(50)',------------------,
to 0.3 mL of 5% infusorial earth (Celite; Fisher Scientific, Springfield, NI) in a pH 7.4 phosphate-buffered saline solution. Calculations necessary to obtain the quantitative porphyrin values were similarly modified:
'E ~ 612(40} >'..J
e-. ... 03459(30)
.c ."
Total urinary porphyrin nmol/L =
[L..J
C, 2.9 100 F. X - X - X F, 0.2 1.08
~~
.~ ~
X15.3,
306(20}
::::>
153(10)
where F. is fluorescence of sample; C" concentration of coproporphyrin standard (0.05 JIg/mL); F" fluorescence of standard; 2.9, final volume ofHCI phase (increased from original 2 mL); 0.2, original volume of urine; 100, conversion factor to 100 mL of extract; and 1.08, conversion factor for the enhancement of porphyrin fluorescence by ethyl acetate saturation of 1.5N HCI; and 15.3, conversion factor from JIg/ dL to nmol/L. This gives total urinary porphyrin measured as nanomoles (nmol) coproporphyrin per liter, understanding that approximately 70% to 80% of total urinary porphyrins in normal individuals are coproporphyrins (I JIg/dL = 15.3 nmol/L).
A 765(~'h---------------_.
c 612(40} '~'J
ti
00
459(30}
[L..J
~~
306(20
::::>
153(10)
~ E °c c
0::-
0
Urinary Creatinine Determinations
o
0
00
B
Urinary creatinine was measured using the Technicon RA System method (Technicon Instruments Corp., Tarrytown, NYf2 based on the Jaffe alkaline picrate reaction.23 The method was automated by Chasson et aV' and optimized by Rossignol et al. 25 Urine samples were diluted fivefold for determinations and results were expressed in micromoles per liter (I mg/dL = 88.4 JImoI/L).
0 0
o .. ..
0
0
0
.. . ..... . 0
o
0
0
0
0
0
00
...... • 0
00
0
4
0 0 0
6
8
10
Age
12
14
16
16
(years)
Fig 1. Total urinary porphyrin as a function of (A) weight and (8) age in pediatric specimens.
Statistical Analysis
to age in children. The direct relationship of values of urinary porphyrin and creatinine in both pediatric and adult specimens is shown in Fig 2. Urinary porphyrin excretion expressed as ~mol/ mol creatinine (or ~g/g) is inversely related to both weight and age in younger children (Fig 3A and B) (I ~gcoproporphyrin/g creatinine = 0.173
Statistical analysis was performed using the Statistical Analysis System programs of SAS Institute, Cary, NC, for linear regression (General Linear Model) and correlation.
RESULTS
Figures 1A and B show total urinary porphyrin concentration (nmol/dL) related to weight and
765(50),~---------------____.
• age ( 18 years fr = 0.72, P ( 0.0001] age.t 18 years r = 0 .77, P ( 0 0001] .
o
612(40)
c
·c
~
>'-1
. .• .. ....••..0
e- ci; 459(30)
£-0
o':~ -I
~ ~ 306(20)
g
.-= :::>
E
c
153( 10)
•
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.0 •
.. "
';.0.0
o
0
•
0
o
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0 .'11
~.01'·
~.t3:.,
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o
o
o
.0
o
o
o
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0
II
0
4.4 8.8 13.3 17.7 22.1 26.5 30.9 35.4 (50) (100) (150) (200) (250) (300) (350) (400)
Urinary Creatinine mmol/L (mg/dL)
Fig 2. Urinary porphyrin as a function of urinary creatinine in pediatric and adult specimens. The solid and dashed lines represent regression lines in the pediatric and adult populations, respectively.
485
URINARY PORPHYRIN EXCRETION c:
86.5(500)-,-----------------,
o~ 0,
~~ 69.2(400) ~~
f~ 51.9(300) ~c ..c r:! \
a.o
°°
E
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~
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, 0
___ ~_
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g[ 17.3(100)
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-~\
0'0 34 .6 (200)
a.
weight) 30 kg [r - -0.12. p = 0.49 (N.S.)] w.ight.!. JO kg [r - -0.59, p (0.0001]
0
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o
00
'00
:
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......
•
10
20
0
•
0
i
30 Wei&ht 50
60
70
BO
(kg)
c:
o~ :;:o~
86.5(500).-------='-------~ ,0
° "',0
~369.2(400) ~~ .§~ 51.9(300)
age = 9 - 18 years [r = -0.30. p = 0.10 (N.S.)] oge ( 9 yeo", [r - -0.61, P (0.0001]
g
-..
E~
a.u
0
0
00 .......
00
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0
':
....
..
0
.........
o-::: .. :::::~ __ -_---
0
0'0 34.6(200), ...... ° 8: o....E 8......: ~~ ...... g Q)cl°o ~ o ~ 17.3(100) ------_:, .§:::s.. -;;:----cr\-o-- _______ :: ....... , 0
0
'
00
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0
0 ..
'.
0
---'"
~
B
o
2
4
6
8
10
Age
12
14
16
18
(years)
Fig 3, Urinary porphyrin excretion expressed as ~moll mol creatinine (~g/g) as a function of (A) weight and (8) age in pediatric specimens. Dashed lines represent 2 SD from solid regression line to which they are parallel.
JLmol/mol). Linear regression analysis was performed on the pediatric data separated into two groups at the point where total urinary porphyrin excretion becomes independent of weight or age (30 kg and 9 years). The urinary porphyrin excretion (JLmol porphyrin/mol creatinine or JLg/g) of patients 9 to 18 years of age was lower than values of younger children (P < 0.0001), but did not significantly differ (P = 0.63) from adult values when not segregated by sex. Pearson correlation coefficients (r) and significance values (P) of the relationships in the pediatric patients of (1) urinary porphyrin concentration and urinary porphyrin excretion to age and weight, (2) urinary creatinine to urinary porphyrins, and (3) creatinine to age and weight are displayed in Table 1. Urinary porphyrins, creatinine, and their excretion ratio are displayed with respect to sex in Figs 4a, b, and c. Dummy variable analysis (Table 2) did not show sex as a statistically significant factor in patients less than 9 years of age. However in the 9- to 18-year-old patients, females had higher urinary creatinine and porphyrin concentrations, but lower excretion of porphyrin per gram creatinine than similarly aged males. Adult men had higher urinary creatinine and porphyrin
Table 1. Correlation Coefficients and Levels of Significance of Measured Variables in Pediatric Patients
Dependent Variable
Urinary creatinine mg/dL
Urinary porphyrin
~g/dL
(~mol/L)
(nmol/L)
Urinary porphyrin excretion creatinine (J'moljmol)
~g/g
* Pearson correlation coefficient.
t P value not significant at 0.05 level.
Independent Variable
No. of Patients
Urinary porphyrin ~g/dL (nmoIfL) Weight <30 kg ;;0,,30 kg Age <9 yr 9-18 yr Weight <30 kg ;;0,,30 kg Age <9 yr 9-18 yr Weight <30 kg ,,;;30 kg Age <9 yr 9-18 yr
r Value"
PValuet
81
0.72
<0.0001
48 33
0.45 0.10
<0.01 0.57t
50 31 81 48 33 81 50 31
0.49 0.15 0.19 0.11 0.01 0.23 0.17 -0.07
<0.001 0.43 0.09t 0.46t 0.95t <0.05 0.25t 0.72t
48 33
-0.59 -0.12
<0.0001 0.49t
48 31
-0.61 -0.30
<0.0001 0.10t
486
BLOOM ET AL
.~
>.~
459(30 )
.c ....
~~
f1..
~
3306(20)
~s.
o
0
.§ ~ :>
153(10I}
o 4)
~~
A
22.1 (2&1)
E: ·c_
:g ~ 17.7(200) ~3
U .... '3.3('50) ~~
~ ~ 8.8(100) .;:: E 4.4 (50) :> c
o
o
B
§
I~
:0%:::
~43.3(250}
~3
ILl ~
c·-
I
34.6(200)
.~~
.cP6.0(150)
e-u
f1..~ 17.3(100)
N .§[ 8.7(50) :>
o
C
~
(9
9 - 18
Age
Adult
(yeors) Fig 4. The effect of age and sex on (A) urinary porphyrin concentration (nmol/L or ",g/dL), (8) urinary creatinine concentration (nmol/L or ",g/dL), and (C) urinary porphyrin excretion (",mol/mol or ",g/g creatinine). All values are expressed as ±SE. D, Males; ~, females.
concentrations but lower porphyrin excretion than adult women. After segregating the data by sex, men had significant increases in urinary creatinine concentration and decreased porphyrin excretion ratios when compared with males 9 to 18 years of age (Table 3). When compared with females 9 to 18 years of age, the higher urinary porphyrin excretion of adult women approached significance (P = 0.05), while the values of their urinary creatinine and porphyrin concentrations were significantly lower (P < 0.00 I for both). DISCUSSION
In previous studies, Hsia and Page found that 24-hour coproporphyrin excretion correlated
better with weight than with age and body surface area in 6- to 16-year-old boys. 1Wolkoff and Arias measured creatinine and coproporphyrin in random urine specimens of 23 premature infants and calculated daily excretion of coproporphyrin by assuming a constant creatinine excretion of 8 mg/kg/24 h. 26 Rocchi et al found healthy infants to have a to-fold decrease in J-Lg coproporphyrin/ g creatinine during the first to days oflife. 27 Other studies indicate that creatinine increases with postnatal age. 28 •29 However, these studies do not provide a uniform assessment of the ranges of normal urinary porphyrin output in children of both sexes and all ages. In comparing our values with those of studies in which high performance liquid chromatography (HPLC) and/or absorption spectrophotometry were used to quantify urinary porphyrins, it should be noted that such methods often yield lesser quantities than those determined by solvent extraction and/or fluorometry.3o Using HPLC, Poh-Fitzpatrick et al found the mean coproporphyrin fraction of total urinary porphyrins in normal children to be at least 70%, with the majority as isomer m.1O We quantified total urinary porphyrins in these normal subjects as coproporphyrin in light of that data, and of data of Enriquez de Salamanca et al placing the mean coproporphyrin fraction of total urinary porphyrin in normal adults near 80%.16 Thus, in comparing our study with any study in which isolated coproporphyrin was quantified, our values would also tend to be higher. The increased concentration values of urinary porphyrin found in adult men are consistent with the higher quantitative coproporphyrinuria or total porphyrinuria found in males by previous investigators. 16,31 The higher concentration of urinary creatinine in men versus women results in lower porphyrin excretion values for men. In the 9- to 18-year-old group, females had higher concentrations of both porphyrin and creatinine, yet lower porphyrin excretion due to the greater magnitude of difference in the creatinine levels. Disparity in urinary creatinine concentrations between males and females in both older age groups probably reflects differences in body mass. Body mass may also be a factor contributing to the similar pattern seen when comparing urinary porphyrin concentrations in males versus females (Fig 4A).
487
URINARY PORPHYRIN EXCRETION Table 2. Effects of Gender and Age Sex Dependent Variable
Urinary creatinine (mg/dL)
Urinary coproporphyrin (/Lg/dL) Urinary prophyrin excretion (/Lg/g creatinine)
Age (yr)
s:9 9-18 >18 s:9 9-18 >18 s:9 9-18 >18
Male (n)t
77.6± 120.4 ± 174.8 ± 13.9 ± 16.8 ± 18.0 ± 209.5 ± 142.6 ± 110.3 ±
, Female (n)t
8.5 (28) 14.8 (16) 15.6 (26) 1.4 (28) 2.1 (16) 1.7 (26) 17.8 (28) 11 .1 (16) 7.7 (26)
89.0 ± 191.5 ± 76.2 ± 14.8 ± 20.2± 9.7 ± 195.5 ± 111.5 ± 142.4 ±
Value:!:
14.3 (19) 24.0 (13) 10.9 (13) 2.0 (19) 2.1 (13) 1.0 (13) 19.1 (19) 9.1 (13) 12.0 (13)
0.11 0 .45 0.57 0.04 0.21 0.49 0 .08 0.37 0.36
PValue§
0.47§ <0.05 <0.001 0.78§ 0.26§ <0.01 0.60§ <0.05 <0.05
, Values are expressed as mean ± SE. "n" denotes sample size. :j: Pearson correlation coefficient. § P value not significant at 0 .05 level.
t
Sanguinetti et al showed the effect of creatinine correction in coproporphyrinuria of workers exposed to lead. 33 Since diurnal variation in urinary porphyrin excretion has been observed in several independent studies, the recommendation that complete 24-hour specimens should still be collected whenever possible seems justified. However, in children or others unable to cooperate, measurement of total porphyrin excretion related to creatinine in random specimens obtained in midafternoon should not fail to detect abnormal coproporphyrinuria, if compared with normal values corresponding to the age, weight, and sex of the individual. Diurnal variation in urinary uroporphyrin and heptacarboxyporphyrin excretion inverse to that of coproporphyrin, with nadirs in the afternoon and higher levels from midnight to noon, was found by Martasek et al in 10 patients with porphyria cutanea tarda. 36 However, in the uropor-
Whether urinary excretion data obtained from random specimens are as useful as those from complete 24-hour specimens depends on the extent of diurnal variation in the metabolites of interest. Circadian rhythm in urinary coproporphyrin excretion, lower during night and morning and maximal in afternoon hours, has been observed in normal adult men 32 and in men with pathologic porphyrinuria,33-34 and can be speculated to result from changes in urinary pH35 or glomerular filtration, reabsorption, or secretion of porphyrins while supine. The values we report herein were from random daytime specimens collected with no attempt to control pH by dietary measures. Since urinary creatinine excretion also decreases during the night and morning and peaks in the afternoon,32,35 expressing porphyrin excretion per unit creatinine would tend to correct diurnal variance, but with greater efficiency in normal or only mildly elevated porphyrinuria.
Table 3. Comparison of Adults and Children (9-18 Years) of Same Gender Dependent Variable
Urinary creatinine mmol/L (mg/dL) Urinary porphyrin nmol/L (!Lg/dL) Urinary porphyrin excretion /Lmol/mol creatinine (!Lg/g)
Sex
, Value'
PValuet
Males Females Males Females Males Females
0.35 -0.67 0 .64 -0.67 -0.36 0.39
<0.05 <0.001 0.08' <0.001 <0.05 0.05t
, Pearson correlation coefficient with positivie values signify increased values of variable in adult population.
t P value not significant at 0.05 level.
488
BLOOM ET AL
phyrinuria in porphyrias occurring in children (porphyria cutanea tarda, congenital erythropoietic porphyria, hepatoerythropoietic porphyria, or other rare homozygous porphyric states), from whom collecting complete 24-hour specimens may be virtually impossible in an ambulatory setting, total porphyrin output is often so high that a falsely normal result by our method would be unlikely, even in a random specimen obtained at a circadian nadir. In such cases, absolute values for total urinary porphyrin as calculated in coproporphyrin units by our simple, rapid, and relatively inexpensive technique would be somewhat in error; however, the amounts of other urinary porphyrins excreted by patients with var-
ious porphyrias are frequently at least an order of magnitude greater than the total porphyrin output of normal adults or children, and would still yield a clearly abnormal result. If abnormal total porphyrinuria is found in random or 24hour specimens, more sophisticated fractionation methods should then be used to determine the relative amounts of porphyrins of different carboxyl number and isomer partition to aid in the determination of the precise diagnosis. ACKNOWLEDGMENT The authors thank Dr Valery Morris and Dr Robert Berk for their help in obtaining clinical specimens, and Jeanine Carr and Dr Gregory Almond for their technical assistance.
REFERENCES I. Hsia DY-Y, Page M: Coproporphyrin studies in children. I. urinary coproporphyrin excretion in normal children. Proc Soc Exp Bioi Med 85:86-88, 1954 2. Ford RE, Ou CN, Ellefson RD: Liquid-chromatographic analysis for urinary porphyrins. Clin Chern 29:527-530, 1981 3. Poh-Fitzpatrick MB, Ramsay CA, Frain-Bell W, et al: Photodermatosis in infants and children. Pediatr Dermatol 5:189-200, 1988 4. Poh-Fitzpatrick MB: Laboratory testing in the porphyrias. Int J Dermatol 18:452-460, 1979 5. Chisolm n, Barrett MB, Mellits ED: Dose-effect and dose-response relationships for lead in children. J Pediatr 87: 1152-1160, 1985 6. Piomelli S: A micromethod for free erythrocyte porphyrins: The FEP test. J Lab Clin Med 81 :932-940, 1973 7. Ostrowski J, Kosecki P, Martynska M, et al: Urinary porphyrins in liver disease. Scand J Gastroenterol 19:862-866, 1984 8. Wolkoff AW, Wolpert E, Pascasio FN, et al: Rotors syndrome: A distinct inheritable pathophysiologic entity. Am J Med 60:173-179, 1976 9. Wolkoff AW, Cohen LE, Arias 1M: Inheritance of the Dubin-Johnson Syndrome. N Eng! J Med 288: 113-117, 1973 10. Poh-Fitzpatrick MB, Zaider E, Sciales C, et al: Cutaneous photosensitivity and coproporphyrin abnormalities in Alagille syndrome. Gastroenterology 99:831-835, 1990 II. Gladen BC, Rogan WJ, Ragan NB, et al: Urinary porphyrins in children exposed transplacentally to polyhalogenated aromatics in Taiwan. Arch Environ Health 43:54-58, 1988 12. Cripps DJ, Peters HA, Gocmen A, et al: Porphyria turcica due to hexachlorobenzene: A 20 to 30 year follow up study on 204 patients. Br J Dermatol III :413-422, 1984 13. Lyberatos M, Papadopoulos N, Papasteriadis E, et al: Urine porphyrins and their percursors in homozygous P-thalassaemia. Acta Haematol 54:95-97, 1975 14. Watson CJ, Schulze W, Hawkinson Y, et al: Copro-
porphyrinuria (type III) in acute poliomyelitis. Proc Soc Exp Bioi Med 64:73-78, 1947 15. Francoual J, Lindenbaum A, Dehan M, et al: Coproporphyrin in urine of newborns with meconium aspiration syndrome. Clin Chern 29:2054-2056, 1983 16. Enriquez de Salamanca R, Pena ML, Chinarro A, et al: Quantitative and qualitative porphyrin excretion in normal subjects. Br J Biochem 14:251-254, 1982 17. Fernandez AA, Henry RJ, Goldenberg H: Assay of urinary porphyrins. Evaluation of extraction methods and choice of instrumentation. Clin Chern 12:463-474, 1966 18. Talman EL: Standard Methods in Clinical Chemistry, vol 2. San Diego, CA, Academic, 1958, pp 137-147 19. Sobel C, Cano C, Thiers RE: Separation and quantitation of coproporphyrin and uroporphyrin in urine. Clin Chern 20: 1397-1402, 1974 20. Green MG (ed): The Harriet Lane Handbook (Johns Hopkins Hospital) (ed 12). Chicago, IL, Year Book, 1991, pp 353-360 21 . Poh-Fitzpatrick MB, Piomelli S, Young P, et al: Rapid quantitative assay for erythrocyte porphyrins. Rapid quantitative microfluorometric assay applicable to the diagnosis of erythropoietic protoporphyria. Arch Dermatol 110:225-230, 1984 22. Technicon Method No. SM4-014IK86. Creatinine. Technicon Instruments Corp., Tarrytown, NY, October 1986 23. Jaffe MZ: Concerning the precipitate produced in normal urine by picric acid and a new reaction of creatinine. Z Physiol Chern 10:391-400, 1986 24. Chasson AI, Grady HJ, Stanley MA: Determinations of creatinine measurement by means of automatic chemical analysis. Am J Clin Pathol 35:83-88, 1961 25. Rossignol B, Rossignol D, PetitClerc C: Improvement of creatinine measurement on RA-IOOO. Clin Biochem 17: 2034, 1984 (abstr) 26. Wolkoff AW, Arias 1M: Coproporphyrin excretion in amniotic fluid and urine from premature infants: A possible maturation defect. Pediatr Res 8:591-593, 1974 27. Rocchi E, Balli F, Gibertini P, et al: Coproporphyrin excretion in healthy newborn babies. J Pediatr Gastroenterol Nutr 3:402-407, 1984
URINARY PORPHYRIN EXCRETION
28. Trompeter LS, aI-Dahhan J, Haycock GB, et aI: Normal values for plasma creatinine concentration related to maturity in normal term and preterm infants. Int J Pediatr Nephrol 4: 145-148, 1983 29. Ross B, Cowett RM, Oh W: Renal functions of low birth weight infants during the first two months oflife. Pediatr Res II : 1162-1164, 1977 30. Gaetani E, Laureri CF, Vitto M: High performance liquid chromatographic differentiation of urinary free porphyrins. J Chromatogr 231 :425-432, 1982 31 . Doss M, Schmidt A: Rapid determination of urinary total porphyrins by ion exchange chromatography. Z K1in Chim Biochem 9:415-418, 1971 32. Araki S, Murata K, Yokoyama K, et a1: Circadian rhythms in the urinary excretion of metals and organic sub-
489 stances in "healthy" men. Arch Environ Health 38:360-366, 1983 33. Sanguinetti F, Dompe M, Mantovani S: Circadian rhythms in the excretion of coproporphyrin and deltaaminolevulinic acid. Ann 1st Super Sanita 14:601-606, 1978 34. Martasek P, Jirsa P, Kordac V: Role of the kidneys in porphyrias. Nephron 32:277-278, 1982 35. Kanabrocki EL, Snedeker PW, Zieher SJ, et aI: Circadian characteristics of dialyzable and non-dialyzable human urinary electrolytes, trace elements and total solids. Chronobiol Int 5:175-184,1988 36. Martasek P, Jirsa M, Kordac V: Diurnal rhythm of excretion of various types of porphyrins in symptomatic hepatic porphyria. Cas Lek Cesk 120:1255-1257, 1981