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Serum Haptoglobins: A Preoperative Detector of Metastatic Renal Carcinoma
Vol. 112, September Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyri~ht © 1974 hy The Williams & Wilkins Co.
SERUM HAPTOGLOBINS: A PREOPERATIVE DETECTOR OF METASTATIC RENAL CARCINOMA MARTYN VICKERS, JR.* From the Division of Urologv, University of Vermont, Burlinfiton, Vermont
pneumonia, tuberculosis), c) an operation, d) obstructive biliary tract disease, e) androgen ingestion and f) temperature elevations.
Haptoglobin is a serum glycoprotein which constitutes 24 per cent of the alpha-2 globulins under basal conditions. 1 Its name, haptos, meaning to bind, reflects its special ability to bind free hemoglobin. Early investigators noted that there were different types of haptoglobins, the individual type being governed by a genome. 2 • 3 Each type is distinguished by its polypeptide makeup and has a half-life of 2 to 3 days. 4 All types are continuously produced in the liver and are filtered from the blood by the reticuloendothelial system. 5 · 7 Each type of haptoglobin possesses its own normal ranges which can be measured with photometric techniques. As a group the various types have a lower limit of20 mg. per cent and an upper limit of 204 mg. per cent under basal conditions. The increased levels of serum haptoglobins may exist in inflammation or infection. 1 The mechanism for elevation is thought to be caused by hepatic production, initiated in an unknown way by the abnormal condition. 8 The response is immediate and persistent and the elevation is proportioned to the intensity of the initiating stimulus. The 3 major limitations to the precision of the haptoglobin test are: 1) daily variations (up to 5 to 7 mg. per cent) may occur in the same individual, 2) reproducibility of laboratory analysis is plus 5 per cent and 3) the coexistence of several conditions capable of elevating serum haptoglobin level: a) collagen vascular diseases (systemic lupus erythematosus), b) acute and chronic infections (lobar
MATERIAL AND METHODS
A prospective study was done on 36 patients between 36 and 86 years old. All patients who were hospitalized between January 1, 1970 and April 1, 1973 for gross or microscopic hematuria, back pain, calcified renal cyst or evidence of metastatic disease were included. 0~ 3 successive preoperative days 5 cc venous blood were obtained for serum haptoglobin determinations. 9 Tissue diagnosis was made by microscopic examination of the resected tissue. Evidence of metastatic spread of the renal carcinoma was obtained by either a review of resected nodes, a positive metastatic survey or autopsy. RESULTS
Accepted for publication January 11, 1974. * Current address: Ireland Army Hospital, Fort Knox, Kentucky 40121. 1 Nyman, M.: Serum haptoglobin: methodological and clinical studies. Scand. J. Clin. Lab. Invest., suppl. :l9, 11: 1, 1959. 2 Smithies, 0. and Walker, N. F.: Notation for serumprotein groups and the genes controlling their inheritance. Nature, 178: 694, 1956. 3 Smithies, 0., Connell, G. E. and Dixon, G. H.: Inheritance of haptoglobin subtypes. Amer. J. Hum. Genet., 14: 14, 1962. 'Giblett, E. R.: Haptoglobin: a review. Vox Sang., 6: 513, 1961. Krauss, S. and Sarcione, E. J.: Synthesis of serum haptoglobin by the isolate perfused rat liver. Biochim. Biophys. Acta, 90: 301, 1964. •Merrill, D. A., Kirkpatrick, C.H., Wilson, W. E. C. and Riley, C. M.: Change in serum haptoglobin type following human liver transplantation. Proc. Soc. Exp. Biol. Med., 116: 748, 1964. 'Wada, T., Ohara, H., Watanabe, K., Kinoshita, H. and Tachi, A.: Auto radiographic study on the site of uptake of the haptoglobin-hemoglobin complex. Reticuloend. Soc., 8: 185, 1970. 8 Ohara, H., Watanabe, K. and Wada, T.: Turnover rate of serum haptoglobin in malignant neoplastic diseases. Clin. Chim. Acta, 19: 41, 1968. 5
310
The 36 patients with abnormal renal masses were reviewed using clinical and radiographic parameters. All 11 patients with benign renal cysts had normal haptoglobin values. Of the 16 patients with renal cancer localized within the renal capsule 14 (87 per cent) had normal preoperative serum haptoglobin levels. One of the 2 exceptions had a coincident acute infection and was rejected from study. Therefore, 14 of 15 patients (94 per cent) had normal haptoglobin levels. All 6 patients with visceral metastases had elevated haptoglobins. The level of serum haptoglobin was independent of the prothrombin time, alkaline phosphatase and also of mass, if the tumor had not extended beyond the kidney. Part A of the figure pictorially summarizes Robson's staging of renal cell carcinoma. Part B of the figure correlates preoperative serum haptoglobin values with Robson's postoperative staging of renal cell carcinoma and illustrates that the more abnormally elevated the haptoglobin the more advanced the stage of the tumor. To further test and emphasize this characteristic we prescribed 2 conditions: 1) in each case the patient must have a renal cell carcinoma but not any coincident conditions which might cause serum haptoglobin level elevations and 2) a normal serum haptoglobin level is indicative of stage 1 disease and an elevated haptoglobin level is indicative of stage 2, 3 or 4 disease. With these conditions we found that 14 of 16 patients 'Javid, ,J. and Horowitz, H. I.: An improved technic for the quantitation of serum haptoglobin. Amer. J. Clin. Path., 34: 35, 1960.
SERUM HAPTOGLOBINS
A
B
202 w
,,
4 w
(9
I I
3
',·
1
~
if)
2
100
200
300
400
500
SERUM HAPTOGLOBIN LEVEL
were correctly assigned stage 1 disease and 7 of 9 patients were correctly stage 2, 3 or 4 disease. A 2-sample Wilcoxon test, adjusted for ties, was used to test the hypothesis that persons scoring abnormally high on blood tests with the aforementioned 2 conditions, would be more likely to be at a high stage (2, 3 or 4) of tumor development than persons who scored normally. The data showed a significant tendency (p less than .001). The Wilcoxon test compares each normal and abnormal pair and scores plus l if abnormally higher and minus 1 if higher. All these numbers are added and adjusted so that the p value can be found in the standard normal table. We have included the Wilcoxon test because of a major deficiency in the test. The chisquare test is designed to test for independence of 2 variables when there is no ordering. In this case there is ordering present since stage 1 is less severe than stage 2. DISCUSSION
Serum haptoglobin has been evaluated in a variety of clinical situations. Abnormal elevations
have been found in 2 basic conditions-inflarnma· tion and infection. In the late 1950s eluded cancer as another condition. After many types of cancer, she concluded that seru.:11 haptoglobin "is thus of no value in the of early or even advanced cancer". Since that early study other have restudied haptoglobins in relation to individual tumors. McFarlane and associates found that successful treatment of Burkitt's lymphoma was reflected decreasing levels of "~,,,~~,, .. 10 M ueller and associates found invaluable in evaluating recurrence of ovarian carcinoma before it was evident physically or Bottinger published a most extensive biochemical aspects of renal carcinoma. Review of his data reveals a significant correlation between metastatic renal carcinoma and increased serurr1 haptoglobins, although this is not discussed in his 10 McFarlane, H., Ngu, V. Osunkoya, B. 0., Luzzatto, L. and""'"""~,,,, C. · Some acute phase proteins in Burkitt lymphoma Nigerians. Clin. Chim. Acta, li7: 325, 1967. 11 Mueller, W. K., Handschumacher, R. and Wade, rvL E.: Serum haptoglobin in patients with ovarian malignancies. Obst. Gynec., 38: 427, 1971.
312
VICKERS
paper. 12 • 13 In 1963 Gordon suggested that "elevation of ... alpha-2 globulins should enhance the suspicion of this diagnosis" (hypernephroma). 14 In 1968 Bowman and Martinez presented 3 cases of hypernephroma and found the common elements of fever, anemia and hyperhaptoglobinemia in each. 15 Finally, in 1970 Utz and associates described 65 patients undergoing nephrectomy for hypernephroma who had hepatic dysfunction syndrome manifested by abnormal values in at least 3 of 5 tests (delayed bromsulphthalein (BSP) retention, hypoprothrombinemia, increased alpha-2 globulin. increased serum bilirubin and increased alkaline phosphatase). They stated, in relation to the alpha-2 globulins, that "9 of 12 patients with normal postoperative values are well (after 9 years) and 4 of the 7 with persistent increase, died". 16 These studies in conjunction with the present study have suggested a statistical interrelationship between metastatic hypernephroma and elevated serum haptoglobin. Several reasons proposed to explain this elevation include mobilization of haptoglobin from extravascular pools, prolongation of the half-life of haptoglobin and increased production of haptoglobin. The first 2 explanations have been excluded by previous studies. The increased production rate of haptoglobin in the presence of neoplasia necessitates the existence of 3 elements-a stimulator, a messenger and a respondent. We already know that the liver is the responding organ but the exact mechanism which causes the excessive production of haptoglobin has not been discovered. Bi:ittinger suggests that the tumor produced an inciting messenger for the liver. Whatever the nature and action of the messenger, not only does it warn the liver of an aggressor but it also quantitates its size. Studies involving tuberculosis, rheumatoid arthritis, infections and Burkitfs lymphoma have shown that the more extensive the disease the higher the haptoglobin level; this has also been true in the present study. There are 3 unique properties of haptoglobin which make it the ideal sentinel system for metastatic disease. First. its response is independent of other liver functions. In all cases in which liver functions were obtained the ESP, prothrombin time, bilirubin and alkaline phosphatase were within normal limits. Moreover, Nyman has shown 12 Bottinger, L. E.: Studies in renal carcinoma. I. Clinical and pathologic anatomical aspects. Acta Med. Scand., 167: 443, 1960. "Bottinger, L. E.: Studies in renal carcinoma. II. Biochemical investigations. Acta Med. Scand., 167: 455, 1960. 14 Gordon, D. A.: The extrarenal manifestations of hypernephroma. Canad. Med. Ass. J., 88: 61, 1963. 15 Bowman, H. and Martinez, E. J .: Fever, anemia, and hyperhaptoglobinemia: an extrarenal triad of hypernephroma. Ann. Intern. Med., 68: 613, 1968. "Utz, D. C., Warren, M. M., Gregg, J. A., Ludwig, J. and Kelalis, P. P.: Reversible hepatic dysfunction associated with hypernephroma. Mayo Clin. Proc., 45: 161, 1970.
that alpha-2 levels cannot be correlated with the aforementioned liver function tests. 1 The second key characteristic of haptoglobin system is quantitative; the degree of serum elevation is proportional to the degree of insult. This has been seen with infections. inflammation and cancer. Given the existence of this sentinel system how can we use it in the evaluation of renal cell carcinoma? First, we can use serum haptoglobin as a determinate variable, that is if the value is elevated then we would be prompted to obtain more elaborate tests (liver, brain and bone scans) in order to exclude metastasis. By these means we would be able to stage renal cell carcinoma more accurately during the preoperative period. This would aid in the evaluation of the indications for and the benefits of surgical, radiational. chemotherapeutic or hormonal treatment. Routine performance of radioisotopic scans in asymptomatic patients with renal cell carcinoma, of course, could be performed with or without the suggestion of an elevated haptoglobin. However, large series of scanning of asymptomatic organs have yielded low positive results. In our study the 10 liver scans were normal and metastatic bone survey yielded no positive findings in the 19 cases studied. Second, by aiding in the accurate staging of the disease, serum haptoglobin might be helpful in prognosticating survival. We do not have sufficient followup on our patients to substantiate this but, accepting the correlation between the serum haptoglobin level and staging and the correlation between staging and survival, we might find that the more abnormally elevated the preoperative haptoglobin the shorter the survival time. Third, serum haptoglobin might be helpful in detecting early recurrence of renal cell carcinoma in patients who were considered an operative cure. We have studied only 3 such patients and the appearance of an elevated haptoglobin in 2 preceded other clinical evidence of recurrence. SUMMARY
Serum haptoglobin levels were obtained preoperatively in 36 patients with abnormal renal masses, to determine the potential of this chemical for predicting the presence of metastases in patients with renal cell carcinoma. Based on these results a correlation of haptoglobin levels and Robson's staging methods for renal cell carcinoma was made. A relationship was found between metastatic renal disease and an elevated serum haptoglobin. Patients with cystic disease of the kidney and encapsulated localized renal tumors had normal serum haptoglobin levels. A measurement of serum haptoglobin levels is suggested as an aid in the preoperative staging of renal cell neoplasms and the detection of metastasis. Drs. Guy Leadbetter, Jr. and M. E. Wade assisted with this study.
THE JOURNAL OF UROLOGY
Copyri~ht © 1974 hy The Williams & Wilkins Co.
SERUM HAPTOGLOBINS: A PREOPERATIVE DETECTOR OF METASTATIC RENAL CARCINOMA MARTYN VICKERS, JR.* From the Division of Urologv, University of Vermont, Burlinfiton, Vermont
pneumonia, tuberculosis), c) an operation, d) obstructive biliary tract disease, e) androgen ingestion and f) temperature elevations.
Haptoglobin is a serum glycoprotein which constitutes 24 per cent of the alpha-2 globulins under basal conditions. 1 Its name, haptos, meaning to bind, reflects its special ability to bind free hemoglobin. Early investigators noted that there were different types of haptoglobins, the individual type being governed by a genome. 2 • 3 Each type is distinguished by its polypeptide makeup and has a half-life of 2 to 3 days. 4 All types are continuously produced in the liver and are filtered from the blood by the reticuloendothelial system. 5 · 7 Each type of haptoglobin possesses its own normal ranges which can be measured with photometric techniques. As a group the various types have a lower limit of20 mg. per cent and an upper limit of 204 mg. per cent under basal conditions. The increased levels of serum haptoglobins may exist in inflammation or infection. 1 The mechanism for elevation is thought to be caused by hepatic production, initiated in an unknown way by the abnormal condition. 8 The response is immediate and persistent and the elevation is proportioned to the intensity of the initiating stimulus. The 3 major limitations to the precision of the haptoglobin test are: 1) daily variations (up to 5 to 7 mg. per cent) may occur in the same individual, 2) reproducibility of laboratory analysis is plus 5 per cent and 3) the coexistence of several conditions capable of elevating serum haptoglobin level: a) collagen vascular diseases (systemic lupus erythematosus), b) acute and chronic infections (lobar
MATERIAL AND METHODS
A prospective study was done on 36 patients between 36 and 86 years old. All patients who were hospitalized between January 1, 1970 and April 1, 1973 for gross or microscopic hematuria, back pain, calcified renal cyst or evidence of metastatic disease were included. 0~ 3 successive preoperative days 5 cc venous blood were obtained for serum haptoglobin determinations. 9 Tissue diagnosis was made by microscopic examination of the resected tissue. Evidence of metastatic spread of the renal carcinoma was obtained by either a review of resected nodes, a positive metastatic survey or autopsy. RESULTS
Accepted for publication January 11, 1974. * Current address: Ireland Army Hospital, Fort Knox, Kentucky 40121. 1 Nyman, M.: Serum haptoglobin: methodological and clinical studies. Scand. J. Clin. Lab. Invest., suppl. :l9, 11: 1, 1959. 2 Smithies, 0. and Walker, N. F.: Notation for serumprotein groups and the genes controlling their inheritance. Nature, 178: 694, 1956. 3 Smithies, 0., Connell, G. E. and Dixon, G. H.: Inheritance of haptoglobin subtypes. Amer. J. Hum. Genet., 14: 14, 1962. 'Giblett, E. R.: Haptoglobin: a review. Vox Sang., 6: 513, 1961. Krauss, S. and Sarcione, E. J.: Synthesis of serum haptoglobin by the isolate perfused rat liver. Biochim. Biophys. Acta, 90: 301, 1964. •Merrill, D. A., Kirkpatrick, C.H., Wilson, W. E. C. and Riley, C. M.: Change in serum haptoglobin type following human liver transplantation. Proc. Soc. Exp. Biol. Med., 116: 748, 1964. 'Wada, T., Ohara, H., Watanabe, K., Kinoshita, H. and Tachi, A.: Auto radiographic study on the site of uptake of the haptoglobin-hemoglobin complex. Reticuloend. Soc., 8: 185, 1970. 8 Ohara, H., Watanabe, K. and Wada, T.: Turnover rate of serum haptoglobin in malignant neoplastic diseases. Clin. Chim. Acta, 19: 41, 1968. 5
310
The 36 patients with abnormal renal masses were reviewed using clinical and radiographic parameters. All 11 patients with benign renal cysts had normal haptoglobin values. Of the 16 patients with renal cancer localized within the renal capsule 14 (87 per cent) had normal preoperative serum haptoglobin levels. One of the 2 exceptions had a coincident acute infection and was rejected from study. Therefore, 14 of 15 patients (94 per cent) had normal haptoglobin levels. All 6 patients with visceral metastases had elevated haptoglobins. The level of serum haptoglobin was independent of the prothrombin time, alkaline phosphatase and also of mass, if the tumor had not extended beyond the kidney. Part A of the figure pictorially summarizes Robson's staging of renal cell carcinoma. Part B of the figure correlates preoperative serum haptoglobin values with Robson's postoperative staging of renal cell carcinoma and illustrates that the more abnormally elevated the haptoglobin the more advanced the stage of the tumor. To further test and emphasize this characteristic we prescribed 2 conditions: 1) in each case the patient must have a renal cell carcinoma but not any coincident conditions which might cause serum haptoglobin level elevations and 2) a normal serum haptoglobin level is indicative of stage 1 disease and an elevated haptoglobin level is indicative of stage 2, 3 or 4 disease. With these conditions we found that 14 of 16 patients 'Javid, ,J. and Horowitz, H. I.: An improved technic for the quantitation of serum haptoglobin. Amer. J. Clin. Path., 34: 35, 1960.
SERUM HAPTOGLOBINS
A
B
202 w
,,
4 w
(9
I I
3
',·
1
~
if)
2
100
200
300
400
500
SERUM HAPTOGLOBIN LEVEL
were correctly assigned stage 1 disease and 7 of 9 patients were correctly stage 2, 3 or 4 disease. A 2-sample Wilcoxon test, adjusted for ties, was used to test the hypothesis that persons scoring abnormally high on blood tests with the aforementioned 2 conditions, would be more likely to be at a high stage (2, 3 or 4) of tumor development than persons who scored normally. The data showed a significant tendency (p less than .001). The Wilcoxon test compares each normal and abnormal pair and scores plus l if abnormally higher and minus 1 if higher. All these numbers are added and adjusted so that the p value can be found in the standard normal table. We have included the Wilcoxon test because of a major deficiency in the test. The chisquare test is designed to test for independence of 2 variables when there is no ordering. In this case there is ordering present since stage 1 is less severe than stage 2. DISCUSSION
Serum haptoglobin has been evaluated in a variety of clinical situations. Abnormal elevations
have been found in 2 basic conditions-inflarnma· tion and infection. In the late 1950s eluded cancer as another condition. After many types of cancer, she concluded that seru.:11 haptoglobin "is thus of no value in the of early or even advanced cancer". Since that early study other have restudied haptoglobins in relation to individual tumors. McFarlane and associates found that successful treatment of Burkitt's lymphoma was reflected decreasing levels of "~,,,~~,, .. 10 M ueller and associates found invaluable in evaluating recurrence of ovarian carcinoma before it was evident physically or Bottinger published a most extensive biochemical aspects of renal carcinoma. Review of his data reveals a significant correlation between metastatic renal carcinoma and increased serurr1 haptoglobins, although this is not discussed in his 10 McFarlane, H., Ngu, V. Osunkoya, B. 0., Luzzatto, L. and""'"""~,,,, C. · Some acute phase proteins in Burkitt lymphoma Nigerians. Clin. Chim. Acta, li7: 325, 1967. 11 Mueller, W. K., Handschumacher, R. and Wade, rvL E.: Serum haptoglobin in patients with ovarian malignancies. Obst. Gynec., 38: 427, 1971.
312
VICKERS
paper. 12 • 13 In 1963 Gordon suggested that "elevation of ... alpha-2 globulins should enhance the suspicion of this diagnosis" (hypernephroma). 14 In 1968 Bowman and Martinez presented 3 cases of hypernephroma and found the common elements of fever, anemia and hyperhaptoglobinemia in each. 15 Finally, in 1970 Utz and associates described 65 patients undergoing nephrectomy for hypernephroma who had hepatic dysfunction syndrome manifested by abnormal values in at least 3 of 5 tests (delayed bromsulphthalein (BSP) retention, hypoprothrombinemia, increased alpha-2 globulin. increased serum bilirubin and increased alkaline phosphatase). They stated, in relation to the alpha-2 globulins, that "9 of 12 patients with normal postoperative values are well (after 9 years) and 4 of the 7 with persistent increase, died". 16 These studies in conjunction with the present study have suggested a statistical interrelationship between metastatic hypernephroma and elevated serum haptoglobin. Several reasons proposed to explain this elevation include mobilization of haptoglobin from extravascular pools, prolongation of the half-life of haptoglobin and increased production of haptoglobin. The first 2 explanations have been excluded by previous studies. The increased production rate of haptoglobin in the presence of neoplasia necessitates the existence of 3 elements-a stimulator, a messenger and a respondent. We already know that the liver is the responding organ but the exact mechanism which causes the excessive production of haptoglobin has not been discovered. Bi:ittinger suggests that the tumor produced an inciting messenger for the liver. Whatever the nature and action of the messenger, not only does it warn the liver of an aggressor but it also quantitates its size. Studies involving tuberculosis, rheumatoid arthritis, infections and Burkitfs lymphoma have shown that the more extensive the disease the higher the haptoglobin level; this has also been true in the present study. There are 3 unique properties of haptoglobin which make it the ideal sentinel system for metastatic disease. First. its response is independent of other liver functions. In all cases in which liver functions were obtained the ESP, prothrombin time, bilirubin and alkaline phosphatase were within normal limits. Moreover, Nyman has shown 12 Bottinger, L. E.: Studies in renal carcinoma. I. Clinical and pathologic anatomical aspects. Acta Med. Scand., 167: 443, 1960. "Bottinger, L. E.: Studies in renal carcinoma. II. Biochemical investigations. Acta Med. Scand., 167: 455, 1960. 14 Gordon, D. A.: The extrarenal manifestations of hypernephroma. Canad. Med. Ass. J., 88: 61, 1963. 15 Bowman, H. and Martinez, E. J .: Fever, anemia, and hyperhaptoglobinemia: an extrarenal triad of hypernephroma. Ann. Intern. Med., 68: 613, 1968. "Utz, D. C., Warren, M. M., Gregg, J. A., Ludwig, J. and Kelalis, P. P.: Reversible hepatic dysfunction associated with hypernephroma. Mayo Clin. Proc., 45: 161, 1970.
that alpha-2 levels cannot be correlated with the aforementioned liver function tests. 1 The second key characteristic of haptoglobin system is quantitative; the degree of serum elevation is proportional to the degree of insult. This has been seen with infections. inflammation and cancer. Given the existence of this sentinel system how can we use it in the evaluation of renal cell carcinoma? First, we can use serum haptoglobin as a determinate variable, that is if the value is elevated then we would be prompted to obtain more elaborate tests (liver, brain and bone scans) in order to exclude metastasis. By these means we would be able to stage renal cell carcinoma more accurately during the preoperative period. This would aid in the evaluation of the indications for and the benefits of surgical, radiational. chemotherapeutic or hormonal treatment. Routine performance of radioisotopic scans in asymptomatic patients with renal cell carcinoma, of course, could be performed with or without the suggestion of an elevated haptoglobin. However, large series of scanning of asymptomatic organs have yielded low positive results. In our study the 10 liver scans were normal and metastatic bone survey yielded no positive findings in the 19 cases studied. Second, by aiding in the accurate staging of the disease, serum haptoglobin might be helpful in prognosticating survival. We do not have sufficient followup on our patients to substantiate this but, accepting the correlation between the serum haptoglobin level and staging and the correlation between staging and survival, we might find that the more abnormally elevated the preoperative haptoglobin the shorter the survival time. Third, serum haptoglobin might be helpful in detecting early recurrence of renal cell carcinoma in patients who were considered an operative cure. We have studied only 3 such patients and the appearance of an elevated haptoglobin in 2 preceded other clinical evidence of recurrence. SUMMARY
Serum haptoglobin levels were obtained preoperatively in 36 patients with abnormal renal masses, to determine the potential of this chemical for predicting the presence of metastases in patients with renal cell carcinoma. Based on these results a correlation of haptoglobin levels and Robson's staging methods for renal cell carcinoma was made. A relationship was found between metastatic renal disease and an elevated serum haptoglobin. Patients with cystic disease of the kidney and encapsulated localized renal tumors had normal serum haptoglobin levels. A measurement of serum haptoglobin levels is suggested as an aid in the preoperative staging of renal cell neoplasms and the detection of metastasis. Drs. Guy Leadbetter, Jr. and M. E. Wade assisted with this study.